Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis

ABSTRACT

The present invention relates to pharmaceutical compositions and a method of inhibiting metastasis using anti-ROR1 antibodies or antigen binding fragments, ROR1 binding peptides and ROR1 vaccines.

RELATED APPLICATION DATA

This application claims the benefit of priority under 35 U.S.C. §119(e)of the U.S. Patent Application No. 61/693,230, filed on Aug. 24, 2012,U.S. Patent Application No. 61/709,055, filed on Oct. 2, 2012 and U.S.Patent Application No. 61/709,803 filed on Oct. 4, 2012, the entirecontents of which are incorporated herein by reference.

GRANT INFORMATION

This invention was made with government support under P01-CA081534 andR37-CA049870 awarded by the National Institutes of Health and DR1-01430California Institute of Regenerative Medicine. The government hascertain rights in the invention.

SEQUENCE LISTING

The material in the accompanying Sequence Listing is hereby incorporatedby reference in its entirety. The accompanying file, named“ST-UCSD3820-1WO_ST25.txt”, was created on Mar. 14, 2013 and is 33 Kb.The file can be accessed using Microsoft Word on a computer that usesWindows OS.

FIELD OF THE INVENTION

The invention relates generally to receptor tyrosine kinase-like orphanreceptor 1 antibodies and vaccines, as well as methods for inhibitingmetastasis.

BACKGROUND INFORMATION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise.Receptor tyrosine kinases (RTKs) play critical roles in celldifferentiation, proliferation, migration, angiogenesis, and survival.The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is anevolutionarily-conserved type I membrane protein that belongs to the RORsubfamily and has extracellular domains that contain immunoglobulin(Ig)-like, Frizzled, and Kringle domains. ROR1-deficient mice display avariety of phenotypic defects within the skeletal and urogenitalsystems, as well as postnatal growth retardation. ROR1 is expressedduring embryogenesis and by a variety of different cancers, but not bynormal post-partum tissues, and can be considered an onco-embryonicsurface antigen. Functional data suggest that ROR1 may function innon-canonical WNT-signaling to promote the survival of malignant cells.More recent studies have shown that non-canonical WNT signaling plays amajor role in basal-like and other subtypes of breast cancer metastasis.Expression of ROR1 human breast cancer is also associated withactivation of the AKT-CREB pathway and enhanced tumor-cell growth.

Receptor-tyrosine kinase like orphan receptor 1 (ROR1) is a conservedembryonic protein whose expression becomes progressively reduced duringembryonic development in mammals. The intact protein, including itsextracellular domain, does not appear to be significantly expressed innormal, adult mammal tissues. In particular, studies have not identifiedsignificant expression of ROR1 on the cell surface of normal adult humantissues, including normal, non-cancerous B cells (Baker et al., Clin.Cancer Res., 14:396 (2008); DaneshManesh et al., Int. J. Cancer,123:1190 (2008) and Fukuda et al., Proc. Nat'l. Acad. Sci. USA, 105:3047(2008)). However, ROR1 is expressed on the cell surface of malignantB-cells (B-CLL) and mantle cell lymphoma (MCL). It has also beenreported that ROR1 is expressed in certain other cancer cell linesincluding Burkett's lymphoma, renal cell carcinoma, colon cancer andbreast cancer (U.S. Patent Application 2007/02075110). Therefore, ROR1can be considered a selective marker for these cancers.

SUMMARY OF THE INVENTION

The invention provides antibodies against ROR1 that can inhibit cancercell growth and metastasis. This invention provides antibodies againstROR1, ROR1 binding peptides and ROR1 peptide vaccines. Further providedare compositions and methods for inhibiting metastasis using anti-ROR1antibodies or antigen binding fragments thereof, ROR1 antibodyimmunoconjugates, ROR1 peptide vaccines or ROR1 binding peptides. In oneembodiment, the invention provides for an isolated anti-human ROR1antibody having the same binding specificity as antibody 99961. In oneaspect, the antibody binds to the Ig-like domain, which is contiguouswith the CRD domain of human ROR-1 (hROR1). In an additional aspect, theantibody binds to an epitope mapping to amino acids 42-160 of hROR-1. Ina further aspect, the antibody binds to an epitope mapping to aminoacids 130-160 of hROR-1. In another aspect, the antibody requires thepresence of glutamic acid at position 138 of hROR-1 for binding.

In an additional embodiment, the invention provides for an isolatedanti-human ROR1 antibody comprising a heavy chain variable region thatis selected from the group consisting of SEQ ID. NO:1, SEQ ID. NO:5, SEQID. NO:9, SEQ ID. NO:13, and SEQ ID. NO:17, and the light chain variableregion is selected from the group consisting of SEQ ID. NO:3, SEQ ID.NO:7, SEQ ID. NO:11, SEQ ID. NO:15 and SEQ ID. NO:19. In one aspect, theantibody according the heavy chain variable region is SEQ ID NO:5 andthe light chain variable region is SEQ ID NO:7.

In one embodiment, the invention provides for an isolated anti-humanROR1 antibody comprising a heavy chain variable region comprised ofCDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID. NO:27,SEQ ID. NO:28, SEQ ID. NO:29, SEQ ID. NO:33, SEQ ID NO:34 and SEQ ID.NO:35, and the light chain variable region comprised of CDR1, CDR2 andCDR3 selected from the group consisting of SEQ ID. NO:30, SEQ ID. NO:31,SEQ ID. NO:32, SEQ ID. NO:36, SEQ ID NO:37 and SEQ ID. NO:38. In oneaspect the a heavy chain variable region comprised of CDR1, CDR2 andCDR3 is comprised of SEQ ID. NO:27, SEQ ID. NO:28 and SEQ ID. NO:29, andthe light chain variable region comprised of CDR1, CDR2 and CDR3selected from the group consisting of SEQ ID. NO:30, SEQ ID. NO:31 andSEQ ID. NO:32

In a further embodiment, the invention provides for an anti-human ROR-1antibody with a binding affinity greater than 41 nM. In an aspect, theantibody binding affinity is between about 500 pM and about 6 nM. In oneaspect, the antibody binding affinity is about 800 pM.

In one aspect, the antibody is 99961 or humanized forms thereof,including antibodies 99961.1, 99961.2, 99961.3 or 99961.4. In anotheraspect, the antibody inhibits metastasis. In an additional aspect, theantibody internalizes and inhibits cell migration. In a further aspect,the antibody internalizes and down modulates vimentin, snail1/2, or ZEB.In a preferred aspect, the antibody is human, humanized, or chimeric.

In another embodiment, the invention provides for a pharmaceuticalformulation comprising the antibody against ROR1 and a pharmaceuticallyacceptable carrier.

A further embodiment provides an isolated nucleic acid encoding theantibody against ROR1. In another embodiment, the invention provides foran expression vector comprising a nucleic acid encoding an antibodyagainst hROR1. In an additional embodiment, the invention provides for ahost cell comprising the nucleic acid encoding an antibody againsthROR1. In a further embodiment, the invention provides for a method ofproducing an anti-human ROR1 antibody comprising culturing the hostcells under conditions to produce the antibody, then optionallyrecovering the antibody.

In one embodiment the invention provides for a vaccine against ROR-1expressing cells, the vaccine comprising a pharmaceutically acceptablecomposition of an isolated or synthetically produced peptide having anamino acid sequence with at least 95% sequence identity to the ROR-1binding region of antibody D10. In one aspect, the amino acid sequenceof the ROR-1 binding region of antibody D10 is VATNGKEVVSSTGVLFVKFGPC.In a further aspect, the amino acid sequence of the ROR-1 binding regionof antibody D10 is EVVSSTGVLFVKFGPC. In another aspect, the ROR-1expressing cell is a cancer cell. In an additional aspect, cancer cellis B cell leukemia, lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon,lung, skin, pancreatic, testicular, bladder, uterine, prostate, oradrenal cancer.

In another embodiment, the invention provides for a vaccine comprising aROR1 binding peptide having an amino acid sequence with at least 95%sequence identity to VATNGKEVVSSTGVLFVKFGPC and a pharmaceuticallyacceptable carrier. In one aspect, the peptide is mammalian. In anadditional aspect, the ROR1 binding peptide is chimeric and/or of human,mouse, rat, porcine, bovine, primate, feline, canine, rabbit, goat,chicken or ursine origin. In another aspect, the vaccine furthercomprises an immunogenic adjuvant. In a further aspect, the adjuvant isan immunogenic carrier moiety conjugated to the binding peptide. In oneaspect, the amino acid sequence of the binding peptide isVATNGKEVVSSTGVLFVKFGPC. In another aspect, the immunogenic carriermoiety is a carrier peptide, such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), ovalbumin, aluminum hydroxide or otherpharmaceutically acceptable immune adjuvant. Examples ofpharmaceutically acceptable immune adjuvants can be found in Methods inMolecular Medicine, Vol. 42: Vaccine adjuvants: Preparation, Methods andResearch Protocols; Edited by D. T. O'Hagan; Humana Press Inc., TotowaN.J. and European Agency for the Evaluation of Medicinal Products,Committee for Proprietary Medicinal Products, Guidelines on Adjuvants inVaccines, London 2004.

In another embodiment, the invention provides for a vaccine comprising aROR1 binding peptide having an amino acid sequence with at least 95%sequence identity to EVVSSTGVLFVKFGPC and a pharmaceutically acceptablecarrier. In an additional aspect, the ROR1 binding peptide is chimericand/or of human, mouse, rat, porcine, bovine, primate, feline, canine,rabbit, goat, chicken or ursine origin. In a further aspect, theadjuvant is an immunogenic carrier moeity conjugated to the bindingpeptide. In one aspect, the amino acid sequence of the binding peptideis VATNGKEVVSSTGVLFVKFGPC. In another aspect, the immunogenic carriermoiety is a carrier peptide, such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA ovalbumin, aluminum hydroxide or otherpharmaceutically acceptable immune adjuvant. Examples ofpharmaceutically acceptable immune adjuvants can be found in Methods inMolecular Medicine, Vol. 42: Vaccine adjuvants: Preparation, Methods andResearch Protocols; Edited by D. T. O'Hagan; Humana Press Inc., TotowaN.J. and European Agency for the Evaluation of Medicinal Products,Committee for Proprietary Medicinal Products, Guidelines on Adjuvants inVaccines, London 2004.

In an additional embodiment, the invention provides for a pharmaceuticalformulation comprising the vaccine comprising a ROR1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC and a pharmaceutically acceptable carrier.

In an additional embodiment, the invention provides for a pharmaceuticalformulation comprising the vaccine comprising a ROR1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC and a pharmaceutically acceptable carrier.

In one embodiment, the invention provides for a ROR1 binding peptidecomprising the amino acid sequence selected from the group consistingof: SEQ ID NO:25 and SEQ ID NO:26. In one aspect, the peptide has anamino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC. In another aspect, the peptide has an amino acidpeptide sequence at least 95% sequence identity to EVVSSTGVLFVKFGPC. Inanother aspect, the binding peptide is mammalian. In an additionalaspect, the binding peptide is chimeric and/or of human, mouse, rat,porcine, bovine, primate, feline, canine, rabbit, goat, chicken orursine origin.

In an embodiment, the invention provides for a pharmaceuticalformulation comprising a ROR1 binding peptide comprising the amino acidsequence selected from the group consisting of: SEQ ID NO:25 and SEQ IDNO:26 and a pharmaceutically acceptable carrier.

In another embodiment, the invention provides for an isolated nucleicacid encoding a ROR1 binding peptide comprising the amino acid sequenceof SEQ ID NO:25 and SEQ ID NO:26. In another embodiment, the inventionprovides for an expression vector comprising the nucleic encoding a ROR1binding peptide comprising the amino acid sequence of SEQ ID NO:25 andSEQ ID NO:26. In a further embodiment, the invention provides for a hostcell comprising the nucleic acid encoding a ROR1 binding peptidecomprising the amino acid sequence of SEQ ID NO:25 and SEQ ID NO:26. Inan additional embodiment, the invention provides for a method ofproducing a peptide comprising culturing the host cell encoding a ROR1binding peptide comprising the amino acid sequence of SEQ ID NO:25 andSEQ ID NO:26 under conditions to produce the binding peptide. In oneaspect, the method to produce a peptide further comprising recoveringthe binding peptide.

In one embodiment, the invention provides for a method of suppressingmetastasis of ROR-1 expressing cancer, the method comprising disruptingepithelial-mesenchymal transition of tumor cells by administering anantibody having the binding specificity of monoclonal antibody 99961, avaccine comprised of a peptide having an amino acid sequence with atleast 95% sequence identity to the ROR-1 binding region of antibody D10or a ROR-1 binding peptide having an amino acid sequence with at least95% sequence identity to VATNGKEVVSSTGVLFVKFGPC. In one aspect, theROR-1 expressing cancer is B cell leukemia, lymphoma, CLL, AML, B-ALL,T-ALL, ovarian, colon, lung, skin, pancreatic, testicular, bladder,uterine, prostate, or adrenal cancer.

In one embodiment, the invention provides for a method of suppressingmetastasis of ROR-1 expressing cancer, the method comprising disruptingepithelial-mesenchymal transition of tumor cells by administering anantibody having the binding specificity of monoclonal antibody 99961, avaccine comprised of a peptide having an amino acid sequence with atleast 95% sequence identity to the ROR-1 binding region of antibody D10or a ROR-1 binding peptide having an amino acid sequence with at least95% sequence identity to EVVSSTGVLFVKFGPC. In one aspect, the ROR-1expressing cancer is B cell leukemia, lymphoma, CLL, AML, B-ALL, T-ALL,ovarian, colon, lung, skin, pancreatic, testicular, bladder, uterine,prostate, or adrenal cancer.

In an additional embodiment, the invention provides a method fortreating or preventing a cancer in a subject, the method comprisingadministering to the subject an antibody having the binding specificityof monoclonal antibody 99961, a vaccine comprised of a peptide having anamino acid sequence with at least 95% sequence identity to the ROR-1binding region of antibody D10 or a ROR-1 binding peptide having anamino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC. In one aspect, the cancer is B cell leukemia,lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, or adrenal cancer.

In an additional embodiment, the invention provides a method fortreating or preventing a cancer in a subject, the method comprisingadministering to the subject an antibody having the binding specificityof monoclonal antibody 99961, a vaccine comprised of a peptide having anamino acid sequence with at least 95% sequence identity to the ROR-1binding region of antibody D10 or a ROR-1 binding peptide having anamino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC. In one aspect, the cancer is B cell leukemia,lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, or adrenal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows high-level expression of ROR1 in breast cancer isassociated with shorter metastasis-free survival and EMT gene signature.(A) The graph was derived from published data available through thePubMed GEO database (GSE2603, GSE5327, GSE2034, and GSE12276).Kaplan-Meier curves depict the prognostic impact of ROR1 expression onoverall metastasis-free survival. For each analysis, 582 cases weresegregated into tertiles with group designated ROR1H representing theone-third of the patients who had tumors with the highest levels of ROR1mRNA, and the group designated ROR1 L representing the one-third ofpatients who had cancers with the lowest levels of ROR1 mRNA. Theone-third of patients who had tumors with intermediate expression ofROR1 mRNA was designated as ROR1M. Metastasis-free survival wasdetermined by Kaplan-Meier analyses, and statistical differences weredetermined by log-rank test. The number of patients in each category,the total metastatic events, and the corresponding P values (chi-squaretest) are shown in the embedded tables. (B) Heat map showing theexpression of ROR1 (top), EMT-related genes (SNAI1 and SNAI2 encodingSnail-1 and Snail-2, ZEB1 encoding ZEB-1, VIM encoding vimentin, CDH2encoding N-Cadherin, CDH1 encoding E-Cadherin, TJP1 encoding ZO-1, TJP3encoding ZO-3, KRT19 encoding CK-19, or CLDN3 encoding Claudin 3, inprimary breast cancer cells isolated from patients. (C) Heat map showingthe expression of EMT-related genes isolated from MDA-MB-231 (left),HS-578T (middle), BT549 (right) cells treated with ROR1-siRNA orCTRL-siRNA. (D) Immunoblots of protein lysates of MDA-MB-231, HS-578T,or BT549 (as indicated on the bottom) transfected with CTRL-shRNA orROR1-shRNA, as indicated at the top. Immunoblots were probed withantibodies specific for the proteins indicated on the left. (E)Immunoblots of protein lysates of MCF7 transfected with a control vectoror a ROR1-expressing vector, as indicated at the top. Immunoblots wereprobed with antibodies specific for the proteins indicated on the left.

FIG. 2 shows expression of ROR1 by breast cancer cell lines isassociated with features of EMT and higher metastatic potential. (A)Morphological changes (40×) of MDA-MB-231, HS-578T, or BT549 (asindicated on the left) transfected with CTRL-shRNA or ROR1-shRNA, asindicated at the top. (B) Expression of CK-19, E-cadherin, or vimentinwere detected by immunofluorescence staining in MDA-MB-231 cellstransfected with CTRL-shRNA or ROR1-shRNA under 63× magnification. (C)Morphological changes (40×) of MCF7 cells transfected with controlvector or ROR1-expressing vector (as indicated at the top). (D)Expression of CK-19, E-cadherin, or vimentin was detected byimmunofluorescence staining of MCF7 cells transfected with eithercontrol vector or ROR1-expression vector (63× magnification). (E) Assaysfor cell migration (left histograms) or invasion (right histograms) onMDA-MB-231, HS-578T, or BT549 transfected with either CTRL-shRNA (black)or ROR1-shRNA (white). All data were normalized to the results of cellstransfected with CTRL-shRNA, which did not differ from those noted forthe parental cell lines. Results are the mean value for each test group(±SEM) (n=3 per test group). (F) Representative photomicrographs ofCTRL-shRNA-transfected MDA-MB-231 (left panels) orROR1-shRNA-transfected MDA-MB-231 (right panels) in assays forcell-migration (top) or invasion (bottom). Data are shown as means±SEM;*P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNA group.

FIG. 3 shows ROR1 silencing reduces breast cancer metastasis aftermammary pad xenograft. (A) Diagram depicting Stage I or II of the study.(B) Tumor volumes over time (days) during stage I. (C) Weight of thetumors excised from each group. (D) Ex vivo photon flux of primarytumors of each group. (E-F) The in vivo (e) lung photon flux or (f)liver photon flux of each mouse during stage II was normalized withprimary-tumor photon flux for each mouse. Histograms depict thenormalized lung and liver photon flux of each group. (G) The in vivolung photon flux during stage II of each group. (H) Horizontal barsindicate mean ex vivo lung photon flux of mice on d21 for each group(left). To the right are representative bioluminescence images of theextirpated lungs from each group. (I) Histograms representlung-weight-index for each group. (J) Representative H&E-stained lungsections. (K) Horizontal bars indicate mean ex vivo liver photon flux ofmice on d21 for each group (left). To the right are representativebioluminescence-images of extirpated livers on d21 of each group. (1)Representative H&E-stained sections of the liver on d21 of mice injectedfor each group. Data are shown as means±SEM. Data are shown asmeans±SEM; P>0.05 is considered not significant (N.S.), *P<0.05,**P<0.01, ***P<0.001, compared with CTRL-shRNA group.

FIG. 4 shows ROR1 silencing reduces experimental pulmonary metastasisand bone metastasis of MDA-MB-231 cell in vivo. A) Kaplan-Meier survivalcurves of mice injected i.v. with 5×105 CTRL-shRNA-transfected orROR1-shRNA-transfected cells (P<0.001 by log-rank test). (B) The in vivolung photon flux of each group over time following injection (left).Representative bioluminescence images of mice from each group aredepicted to the right. (C-E) Representative H&E-stained sections of thelung on (c) d3, (d) d21, and (e) d28. (F) Bottom histograms provide exvivo lung GFP photon flux on d28 for each group. Representativebioluminescence images of the lungs extirpated on d28. (G) Thelung-weight-index from each group on d28 (bottom). Representativephotographs of the lungs (top) of each group. (H) Kaplan-Meier survivalcurves of mice injected i.c. with 1×105 CTRL-shRNA-transfected orROR1-shRNA-transfected cells (P=0.0017 by log-rank test). (I)Representative bioluminescence images of mice following i.c. tumorinjection. The white boxes define the area from which we acquired thebioluminescence data presented in (j). (J) The histograms provide thenormalized in vivo bone photon flux of each group. (K) The ex vivo bonephoton flux of the extracted pelvic bones of each group on d21.Representative bioluminescence images of the extracted pelvic bones aredepicted to the right. (L) Representative H&E-stained histological bonesections of mice from each. Mouse cartoon is modified from reference(30). Data are shown as the means±SEM *P<0.05, **P<0.01, ***P<0.001,compared with CTRL-shRNA group.

FIG. 5 shows an anti-ROR1 antibody reduces pulmonary metastasis ofMDA-MB-231 cell in vivo. (A) D10 mAb causes internalization of ROR1.MDA-MB-231 cells were stained with control-IgG-Alexa647 (red), orD10-Alexa647 for 30 min on ice, and then either kept on ice (blue) ortransferred to 37° C. for 1 h (orange) prior to flow cytometry. (B)Confocal microscopy of D10-stained (green) MDA-MB-231 cells before andafter 1 h incubation at 37° C. (C) MDA-MB-231 cells were treated with orwithout (-) control IgG (IgG) or D10 for 24h prior to staining with afluorochrome-labeled, non-cross-blocking anti-ROR1, without loss inviability. Mean fluorescence intensity (MFI) of treated cells is shown(***P<0.001 by One-way ANOVA). (D) Representative Immunoblots probed forvimentin (top) or β-actin (bottom) of lysates prepared from MDA-MB-231before (Oh) or after 1, 4, or 24 h treatment with D10 or control IgG.The ratios of vimentin to β-actin band-intensity are provided below. (E)Immunoprecipitates of MDA-MB-231 cell-lysate using control IgG (IgG) oranti-ROR1 (ROR1) were used for immunoblot analyses probed withantibodies specific for vimentin (top) or ROR1 (bottom). (F) Histogramsprovide the number of migrated MDA-MB-231 cells that were pre-treatedfor 1 h with D10 or control IgG. (G) Left, histograms depicting the invivo lung photon flux. Right, Representative H&E-stained sections of thelungs. (H) The graph depicts the normalized in vivo lung photon flux.(I) Representative bioluminescence images of tumor-injected mice treatedwith IgG (top) or D10 (bottom). (J) The histogram depicts thelung-weight-index. (K) Representative H&E-stained sections of the lungs.Data are shown as means±SEM; *P<0.05, **P<0.01, ***P<0.001, comparedwith IgG group.

FIG. 6 shows the chimeric constructs used to map the epitope of ROR1antibody D10. The light portion of the construct is mouse and the darkerportion is human.

FIG. 7 depicts epitope mapping for the D10 antibody, which does notreact with mouse ROR1 protein. The mouse or human ROR1 protein have thedifferent amino acid residues at amino acid positions 138, 142, or 160;the human ROR1 protein has amino acid residues E, S, or Y, at thesepositions, whereas the mouse ROR1 protein has amino acid residues K, T,or S at amino acid positions 138, 142, or 160, respectively. Wegenerated recombinant human ROR1 proteins having either the mouse orhuman amino acid residue at these positions only. These recombinantproteins were separated in non-denaturing polyacrylamide gel and thentransferred onto nylon, which was probed with the D10 mAb. As can beseen in this figure, D10 reacts with recombinant proteins 1, 3, 4, and7, but not 2, 5, or 6, which are described in the legend below. Notethat substitution of the human amino acid residue E at position 138 ofthe human ROR1 protein with the mouse amino acid residue T at position138 abrogates D10 binding.

FIG. 8 shows epitope mapping for the anti-human ROR1 antibody 4A5. Themouse or human ROR1 protein have the different amino acid residues atamino acid positions 88, 105, 109, or 111; the human ROR1 protein hasamino acid residues T, L, S, or I at these positions, whereas the mouseROR1 protein has amino acid residues S, I, A, or N at amino acidpositions 88, 105, 109, or 111, respectively. We generated recombinanthuman ROR1 proteins having either the mouse or human amino acid residueat these positions only. These recombinant proteins were separated innon-denaturing polyacrylamide gel and then transferred onto nylon, whichwas probed with the 4A5 mAb. As can be seen in this figure, 4A5 couldbind to recombinant proteins 1, 2, 3, or 5, but not 4. Recombinantprotein 4 is the human ROR1 protein but with the mouse amino acidresidue N at position 111 instead of the amino acid residue I, which isfound in the human ROR1 protein.

FIG. 9 demonstrates that anti-human ROR1 antibody D10 inhibitsmetastasis of breast cancer cells. A-B. The D10 monoclonal antibodyfacilitates ROR1 receptor internalization. C. 24 hours anti-ROR1antibody D10 treatment decrease ROR1 surface expression in MDA-MB-231cells. D. ROR1 forms complex with vimentin in breast cancer MDA-MB-231cells. E. D10 antibody treatment in vitro could decrease vimentinexpression. F. Anti-human ROR1 antibodies decrease breast cancermigration in vitro. G. The D10 monoclonal antibody inhibits MDA-MB-231breast cancer early-stage (day 2) lung metastasis. H. The D10 monoclonalantibody inhibits MDA-MB-231 breast cancer lung metastasis. I.Representative mice injected with 5E5 MDA-MB-231 cells are shown in thedorsal position. J. Anti-human ROR1 antibody treatment reduced the lungweight of MDA-MB-231-bearing mice. K. Representative pulmonary H&Ehistology from MDA-MB-231-bearing mice after anti-ROR1 antibodytreatment. The error bars indicate SEM; *p<0.05, **p<0.01; based on aunpaired two-sided student's t-test.

FIG. 10 depicts high affinity antibodies generated against the ROR1epitope recognized by mAbs D10, 99451, 99961, or 99221. The mouse orhuman ROR1 protein have the different amino acid residues at amino acidpositions 138, 142, or 160; the human ROR1 protein has amino acidresidues E, S, or Y, at these positions, whereas the mouse ROR1 proteinhas amino acid residues K, T, or S at amino acid positions 138, 142, or160, respectively. We generated recombinant human ROR1 proteins havingeither the mouse or human amino acid residue at these positions only.These recombinant proteins were separated in non-denaturingpolyacrylamide gel and then transferred onto nylon, which was probedwith the each of the three mAb, 99451, 99961, or 99221, as indicated onthe left margin. As can be seen in this figure, each of these antibodiesreacts with recombinant proteins 2, 4, 5, and 8, but not 2, 3, 6, or 7,which are described in the legend below. Note that substitution of thehuman amino acid residue E at position 138 of the human ROR1 proteinwith the mouse amino acid residue T at position 138 abrogates thebinding of either 99451, 99961, or 99221.

FIG. 11 depicts the binding activity of antibodies D10 or 99961 forwild-type or recombinant ROR1 protein. Vectors encoding the human orchimeric ROR1 protein were transfected into 293 cells. This allowed forproduction of recombinant human-mouse chimeric ROR1 protein that thencould be size separated in a non-denaturing PAGE gel (right) or SDS-PAGEgel (left) for immunoblot analysis with different anti-ROR1 mAb. Theresults indicate that both D10 and 99961 antibodies bind to the sameregion, on the C-terminus of Ig-like domain, and that D10 and 99961 canbind to ROR1 under both denatured and native conditions. Note that D10and 99961 bind to all recombinant proteins except for 13. The #13chimeric protein is as described in FIG. 6. The full human extracellulardomain is provided on the far left lane of either gel.

FIG. 12 shows characterization of anti-human ROR1 antibody 99961. A, B.the 99961 antibody was able to block CLL engraftment in transgenic mice.C. the 99961 antibody has a binding affinity approximately 50× greaterthan the D10 antibody.

FIG. 13 shows the specific activity of 99961 against CLL cells in humancord blood reconstituted immune deficient mice. A. 99961 antibodyeliminates >90% of CLL cells. B, C. 99961 antibody has no effect onnormal B or T cell development.

FIG. 14 shows the specific activity of 99961 in ROR+ primary AML.

FIG. 15 shows that the epitope recognized by 99961 is not expressed bynormal hematopoietic stem or progenitor cells.

FIG. 16 shows that 99961 does not cross react with normal adult tissue.

FIG. 17 shows PK studies on 99961 in immune deficient mice.

FIG. 18 illustrates design of a ROR1 peptide vaccine. Three differentantibody epitopes were used to make peptides A19, R22 and K19. Above thebars that correspond to either the human (top) or mouse (bottom) ROR1,are bars labeled A19, R22, or K19. These bars describe the location ofthe peptides, A19, R22, or K19 in the ROR1 extracellular domain.

FIG. 19 shows the method used to conjugate KLH to the peptides.

FIG. 20 shows the peptide design of peptide R22. A cysteine was added tothe C-terminal of the peptide to be used to conjugate to KLH.

FIG. 21 shows that that D10 and 99961 bind to the R22 peptide while 4A5does not.

FIG. 22 shows the immunization scheme for R22 immunization of BALB/cmice.

FIG. 23 shows immunoblot analysis of the epitope that the R22 inducedROR1 antibodies bind on ROR1.

FIG. 24 shows the immunization scheme for R22-KLH in C57BL/6 mice.

FIG. 25 shows FACS analysis of ROR1-positive MDA-MB-231 breast cancercells that had been incubated with anti-R22-KLH antisera at 4° C. or 37°C. for 1 h and then counter-stained with isotype-control-Alexa647-labelsantibody, or 4A5-Alexa647 conjugate for 30 min on ice prior to FACSanalysis of ROR1 expression. The results showed that anti-ROR1 sera fromtransgenic mice induced ROR1 receptor internalization at 37° C., but notat 4° C.

FIG. 26 shows anti-ROR1 sera from transgenic mice immunized with R22-KLHinhibits breast cancer migration in vitro.

FIG. 27 shows the immunization scheme for R-22-KLH in C57BL/6 mice.

FIG. 28 shows the titration curves of antisera of mice immunized withKLH conjugates of any one of the three peptides described in FIG. 18.Depicted is the antisera binding to polystyrene plates coated with humanROR1 protein as assessed via ELISA.

FIG. 29 shows FACS analysis of EW36, JeKo-1, or CLL cells. For thisstudy, a dilution of antisera from mice immunized with R22-KLH wasincubated with the cells for 20 minutes at 4 degrees C. The cells thenwere washed and then labeled with a goat anti-mouse Ig that wasconjugated with a fluorochrome for detection by flow cytometry. The openhistograms are the cells stained with the goat anti-mouse Ig withoutfirst incubating the cells with the R22-KLH antisera. The shadedhistograms are the fluorescence of cells that first were incubated withthe anti-R22-KLH antisera. The increase in fluorescence of the cells isdue to the mouse anti-ROR1 antibodies bound to the surface, which thenwere detected with the goat anti-mouse Ig. The pre-immunization antiseraof these mice or the antisera of mice immunized with KLH did not bind tothese cells.

FIG. 30—The cells indicated in the legend were washed and plated at 25μl with 5×10⁵ cells per well in RPMI/10% FBS in round-bottom 96-wellplates (Corning Costar). The diluted antisera (25 μl) and 25 μl of a 1:5dilution of baby rabbit complement were added per well. D10 mAb was usedas a positive control. All conditions were performed in triplicate.Plates were incubated for 4h at 37° C., and cells were immediatelyquantitated for viability by DiOC6/PI staining and Flow CytometricAnalysis. This study indicates that either D10 or the antisera generatedagainst the R22-KLH peptide could direct complement-mediated lysis ofcells bearing human ROR1. Cells that did not bear ROR1 were not killed(not shown).

FIG. 31 shows the first R22-KLH immunization scheme for C57BL/6 mice.This peptide was conjugated with Keyhole limpet hemocyanin (KLH) andthen used to immunize C57BL/6 mice according to the schema illustratedabove. The first injection of KLH or R22-KLH was in complete Freund'sadjuvant (CFA). The second and subsequent injections were in incompleteFreund's adjuvant (IFA). The animals were bled on the days marked withthe purple arrow. 44 days after the day of the first injection, theC57BL/6 mice were challenged with human-ROR1-expressing CLL thatoriginated in a human ROR1-transgenic C57BL/6 mouse that also wastransgenic for the T-cell-leukemia 1 (TCL1 gene), also under the controlof a B-cell specific promoter/enhancer (E-Cμ). This leukemia resembleshuman CLL and expresses human surface ROR1.

FIG. 32 shows the results of immunization with R22-KLH. A. Arepresentative spleen from mice immunized with KLH versus a mouseimmunized with R22-KLH. B. Inhibition of Engraftment of ROR1+ CLL byimmunization with ROR1 peptide R22-KLH in C57BL/6 mice.

FIG. 33 shows the second immunization scheme for the R22-KLH in C57BL/6mice.

FIG. 34 shows the results of immunization with R22 peptide. A. Spleensfrom a mouse immunized with KLH and a mouse immunized with R22-KLH. BInhibition of Engraftment of ROR1+ CLL following immunization withR22-KLH in C57BL/6 mice.

FIG. 35 is FACS analyses of splenocytes from C57BL/6 mice immunized witheither KLH (top row) or R22-KLH (bottom row), usingflurochrome-conjugated mAb specific for B220 (y-axis) or ROR1 (x-axis).The mAb used to stain the cells binds to a non-crossblocking epitope ofROR1 than the antibodies induced by R22-KLH. The box delineates the areain which the leukemia cells are detected. Note that there are muchfewer, if any, leukemia cells in the spleens of mice immunized with theR22-KLH vaccine.

FIG. 36 is FACS analysis of ROR1 on the ROR1+ CLL cells, which indicatesthat ROR1 was down-modulated after immunization with R22-KLH in C57BL/6mice.

FIG. 37 is FACS analysis of CD8+ T cells present in mice that wereimmunized with KLH or R22-KLH. A. Immunization with R22 causes anincrease in the number of CD8+ T cells, which was absent in miceimmunized with KLH. The bottom panel shows the percentage of CD8+ Tcells from the spleens of mice first immunized 75 days earlier witheither KLH, or R22-KLH.

FIG. 38 shows the immunization scheme for R22-KLH immunization of ROR1transgenic mice.

FIG. 39 is FACS analysis of the inhibition of ROR+ CLL engraftment byimmunization with ROR1 peptide R22 in ROR1-Tg mice.

FIG. 40 shows the results of immunization with R22-KLH in ROR1transgenic mice. ROR1+ CLL was inhibited following immunization withR22-KLH in ROR1 transgenic mice.

FIG. 41 is FACS analysis of ROR1 on the ROR1+ CLL cells, which indicatesthat ROR1 was down-modulated after immunization with R22-KLH in ROR1transgenic mice.

FIG. 42 is FACS analysis of CD3+ T lymphocytes present in ROR1-Tg micethat were immunized with KLH or R22-KLH. Panel A shows that immunizationwith R22-KLH caused an proliferation of T lymphocytes. Panel B shows thepercentage of CD3+ T lymphocytes harvested from the spleens of mice onday 75.

FIG. 43 is FACS analysis of CD4+ T cell present in mice that wereimmunized with KLH or R22-KLH. Panel A shows that immunization withR22-KLH causes an increase in the number of CD4+ T cells, which notdetected in mice immunized with KLH. Panel B. Shows the percentage ofCD4+ T cells harvested from the spleens of mice on day 75.

FIG. 44 is FACS analysis of CD8+ T cell present in mice that wereimmunized with KLH or R22-KLH. Panel A shows that immunization withR22-KLH causes an increase in the number of CD8+ T cells, which notdetected in mice immunized with KLH. Panel B. Shows the percentage ofCD8+ T cells harvested from the spleens of mice on day 75.

FIG. 45 shows high-level expression of ROR1 in breast cancer isassociated with shorter lung, bone and brain metastasis-free survival.The graph was derived from published data available through the PubMedGEO database (GSE2603, GSE5327, GSE2034, and GSE12276). Kaplan-Meiercurves depict the prognostic impact of ROR1 expression on (A) lungmetastasis-free survival, (B) bone metastasis-free survival, or (C)brain metastasis-free survival. For each analysis, 582 cases weresegregated into tertiles with group designated ROR1H representing theone-third of the patients who had tumors with the highest levels of ROR1mRNA, and the group designated ROR1L representing the one-third ofpatients who had cancers with the lowest levels of ROR1 mRNA. Theone-third of patients who had tumors with intermediate expression ofROR1 mRNA was designated as ROR1M. Metastasis-free survival wasdetermined by Kaplan-Meier analyses, and statistical differences weredetermined by log-rank test. The number of patients in each category,the total metastatic events, and the corresponding P values (chi-squaretest) are shown in the embedded tables.

FIG. 46 shows high-level expression of ROR1 in breast cancer isassociated with shorter metastasis-free survival, and independent fromtheir ER, PR and HER2 status. Cohort of 582 patients with breastadenocarcinoma were included in the survival analysis. (A) Comparison ofthe levels of ROR1 mRNA expression of the malignant cells of ERNeg(n=242) and ER+(n=325) breast cancer patients (left panel), PRNeg(n=274) and PR+(n=271) breast cancer patients (center panel), andHER2Neg (n=404) and HER2+(n=106) breast cancer patients (right panel).Results are means±SEM The p value was determined by Student's t-test.(B) Prognostic impact of ER status on overall-metastasis-free survival(P=0.13 by log-rank test). (C) Prognostic impact of ER status and ROR1mRNA expression on overall-metastasis-free survival (P<0.0001 bylog-rank test). (D) PR status on overall-metastasis-free survival(P=0.0007 by log-rank test). (E) Prognostic impact of PR status and ROR1mRNA expression on overall metastasis-free survival (P<0.0001 bylog-rank test). (F) HER2 status on overall-metastasis-free survival(P=0.16 by log-rank test). (G) Prognostic impact of HER2 status and ROR1mRNA expression on overall metastasis-free survival (P<0.0001 bylog-rank test).

FIG. 47 shows expression of ROR1 by breast cancer cell lines isassociated with features of EMT. (A) Immunoblots of lysates fromMDA-MB-231 transfected with CTRL-shRNA or ROR1-shRNA were probed withantibodies specific for ROR1 (top) or (3-actin (bottom) as indicated onthe left. (B) Mean amount of VIM and KRT19 (±SEM), as detected viaqRT-PCR on triplicate samples. Data are shown as means±SEM; *P<0.05,**P<0.01, compared with CTRL-shRNA group.

FIG. 48 shows silencing ROR1 reduces expression of CXCR4. (A) Histogramsindicating the amount of CXCR4 mRNA detected via qRT-PCR in triplicatesamples of MDA-MB-231 transfected with either CTRL-shRNA2 orROR1-shRNA2, as indicated at the bottom of each histogram. (B)Representative flow cytometry fluorescence histograms of ROR1-shRNA2(open histogram with green line) or CTRL-shRNA2 (open histogram withblue line) transduced MDA-MB-231 cells stained with anti-CXCR4-APC mAbor isotype-control mAb (shaded histograms), respectively. (C) Cells wereseeded into the top chambers of trans-wells without BD Matrigel™ toexamine for chemotaxis to CXCL12, which added to a final concentrationof 200ng/m1 to the bottom chambers. The cells that migrated aftersix-hours at 37° C. were enumerated under 10× magnification. Thehistograms each provides the numbers of migrated cells in each of threechambers seeded with MDA-MB-231 cells transfected either with CNTL-shRNAor ROR1-shRNA, as indicated at the bottom of the histogram. Results arerepresentative of 3 independent experiments. Data are shown asmeans±SEM; *P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNAgroup.

FIG. 49 shows silencing ROR1 regulates EMT genes expression. Histogramsindicating the relative mRNA amount of variety genes, as indicated atthe bottom of each histogram, detected via qRT-PCR in triplicate samplesof MDA-MB-231(A), HS578T(B), and BT549(C) transfected with eitherCTRL-siRNA or ROR1-siRNA. Results are representative of 2 independentexperiments. Data are shown as means±SEM; *P<0.05, **P<0.01, comparedwith CTRL-siRNA group.

FIG. 50 shows silencing ROR1 effects modest late-growth inhibition oforthotopic xenografts at the site of injection but strong inhibition ofexperimental pulmonary metastases. (A) RAG-/-γc-/- mice were givensubcutaneous (s.c.) or intravenous (i.v.) injections ofCTRL-shRNA-transfected or ROR1-shRNA-transfected MDA-MB-231. Thebioluminescence photon flux of the primary tumor in the injected mammaryfat pad or of the lung of each mouse was normalized against the photonflux detected for the first measurement following the injection of tumor(100 represents 100% of the photon flux detected on the day of theinitial measurement) (top panels). The top three graphs depict thenormalized bioluminescence photo flux of the mammary fat pads of micegiven s.c. injections of 1×106 (left), 5×105 (center), or 2.5×105(right) indicated cells. The bottom graphs provide normalizedbioluminescence photo flux of the lung of mice given i.v. injections of1×106 (left), 5×105 (center), or 2.5×105 (right) indicated cells. (note:the bottom left graph depicts the actual mean bioluminescence photonflux of the lungs of mice given i.v. injections of 1×106 indicatedcells. (B) The histograms depict the lung-weight-index for mice of eachgroup on d21 (n=5-8) i.v. injected with CTRL-shRNA-transfected (black)or ROR1-shRNA-transfected MDA-MB-231 (grey) or no cells (white). The Pvalues were determined by One-way ANOVA. (C) H&E-stained sections of thelung representative of mice from each group on d21. Data are shown asmeans±SEM *P<0.05, **P<0.01, ***P<0.001, compared with CTRL-shRNA group.

FIG. 51 shows immunohistochemistry of experimental metastatic foci.RAG-/-γ-c-/- mice were given intravenous (i.v.) injections of 5×105CTRL-shRNA-transfected MDA-MB-231 (top panels) or ROR1-shRNA-transfectedMDA-MB-231 (bottom panels). (A) Sections of lung were prepared fromanimals euthanized on day 21. The lungs of mice injected withROR1-shRNA-transfected cells had few metastatic foci, which wereidentified for immunohistochemistry analysis. The sections were stainedwith mAbs specific for Ki67+, CK-19, or vimentin, or terminaldeoxynucleotidyl transferase dUTP nick end labeling (Tunnel). (40×magnification). (B) Sections of lung as in (a) were stained with mAbspecific for phospho-AKT (left panel) or phospho-CREB (right panel) (40×magnification).

FIG. 52 shows silencing ROR1 reduces pulmonary metastasis and bonemetastasis of MDA-MB-231 derived cell lines LM2-4175 and BoM-1833 invivo. (A) Schematic diagram showing that LM2-4175 cells preferentiallymetastasize to lung and BoM-1833 cells preferentially metastasize tobone. Flow cytometry analyses showing the ROR1 expression in LM2-4175and BoM-1833. Mouse cartoons are modified from reference (Cancer Cell,2009; 1; 67-78) (B-C) Flow cytometry analyses showing the ROR1 silencingefficiency in LM2-4175 and BoM-1833, using ROR1-shRNA2. (D) Mice wereeach given an i.v. injection of 2×105 CTRL-shRNA-transfected orROR1-shRNA-transfected LM2-4175 cells. Left, representativebioluminescence images of each group; Right, normalized in vivo lungphoton flux of each group. (E) Kaplan-Meier survival curves of miceinjected i.v. with 2×105 indicated LM2-4175 cells (P<0.0001 by log-ranktest). (F) The lung-weight-index of each group on d21 (bottom).Representative photos of the lungs of each group (top). (G) The ex vivolung GFP photon flux of each group on d21 (bottom). Representativephotos of the bones of each group (top). (H) Representative H&E-stainedhistological sections of the lung on d21. (I) Mice were each given ani.c. injected of 1×105 CTRL-shRNA-transfected or ROR1-shRNA-transfectedBoM-1833 cells. Top, representative bioluminescence images of eachgroup; Bottom, normalized in vivo bone photon flux of each group. (J)Representative bone ex vivo photon flux and H&E-stained histologicalsections of the bone on d21. (K) Representative liver ex vivo photonflux and H&E-stained histological sections of the liver on d21. Data areshown as means±SEM; *P<0.05, **P<0.01, ***P<0.001, compared withCTRL-shRNA group.

FIG. 53 shows silencing ROR1 inhibits migration of HS-578T and BT549 invitro. Data are shown as the means±SEM *P<0.05, **P<0.01, ***P<0.001,compared with cells treated with control IgG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the seminal discovery of compositionsand methods of inhibiting metastasis using anti-ROR1 antibodies orantigen binding fragments thereof, ROR1 antibody immunoconjugates, ROR1peptide vaccines or ROR1 binding peptides.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

ROR1

Applicants have previously discovered expression of full-length ROR1 innumerous cancer cell lines and samples, but not other tissues, includingblood or splenic lymphocytes of non-leukemic patients or normal adultdonors, and also generated mouse anti-sera against full-length humanROR1. Fukuda et al., Blood: ASH Annual Meeting Abstracts 2004 104,Abstract 772 (2004) (incorporated herein by reference in its entirety).The polypeptide and coding sequences for ROR1 have been reportedelsewhere and are also incorporated herein by this reference (see, e.g.,Accession Nos. NP_(—)005003.1 and NM 005012.1). Cancer cells thatexpress the Wnt5a protein, such as CLL cells, not only bind ROR1 buthave a survival advantage conferred as a consequence. The inventiontherefore provides means to utilize the specificity of ROR-1 expressionin cancer cells to treat or prevent cancer.

It has been shown that ROR1 expression enhances resistance to apoptosisand promotes cancer cell growth. As shown in the examples, expression ofROR1 associates with the epithelial-mesenchymal transition (EMT), whichoccurs during embryogenesis and cancer metastasis. High-level expressionof ROR1 associates with enhanced rates of relapse and metastasis inpatients with breast adenocarcinoma. Silencing ROR1 in metastasis-pronebreast-cancer cell-lines attenuated expression of EMT-associatedproteins (e.g. Vimentin, Snail-1/2, and ZEB), enhanced expression ofepithelial cytokeratins and tight junction proteins (e.g. CK-19 andZO-1), and impaired their migration/invasion capacity and metastaticpotential. Treatment of MDA-MB-231 with D10, a mAb specific for ROR1,down-modulate vimentin (which associates with ROR1) to inhibitcancer-cell migration. Administration of D10 to immune-deficient miceengrafted with MDA-MB-231 significantly inhibits tumor metastasis.

Antibodies

Certain embodiments comprise immunopeptides directed against the humanROR1 protein. The immunoglobulin peptides, or antibodies, describedherein are shown to bind to the ROR1 protein. The ROR1 binding activityis specific; the observed binding of antibody to ROR1 is notsubstantially blocked by non-specific reagents. These ROR1 specificantibodies can be used to differentiate between ROR1 cells and normalcells. The ROR1 specific antibodies can also be used in immunotherapyagainst a ROR1 cancer, to determine the response after therapy for aROR-1 cancer and to inhibit metastasis. Such immunopeptides can beraised in a variety of means known to the art

As used herein, the term antibody encompasses all types of antibodiesand antibody fragments, e.g., polyclonal, monoclonal, and those producedby the phage display methodology. Particularly preferred antibodies ofthe invention are antibodies that have a relatively high degree ofaffinity for ROR1. In certain embodiments, the antibodies exhibit anaffinity for ROR1 of about Kd<10⁻⁸ M.

Substantially purified generally refers to a composition which isessentially free of other cellular components with which the antibodiesare associated in a non-purified, e.g., native state or environment.Purified antibody is generally in a homogeneous state, although it canbe in either in a dry state or in an aqueous solution. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography.

Substantially purified ROR-1-specific antibody will usually comprisemore than 80% of all macromolecular species present in a preparationprior to admixture or formulation of the antibody with a pharmaceuticalcarrier, excipient, adjuvant, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other co-ingredient. Moretypically, the antibody is purified to represent greater than 90% of allproteins present in a purified preparation. In specific embodiments, theantibody is purified to greater than 95% purity or may be essentiallyhomogeneous wherein other macromolecular species are not detectable byconventional techniques.

Immunoglobulin peptides include, for example, polyclonal antibodies,monoclonal antibodies, and antibody fragments. The following describesgeneration of immunoglobulin peptides, specifically ROR1 antibodies, viamethods that can be used by those skilled in the art to make othersuitable immunoglobulin peptides having similar affinity and specificitywhich are functionally equivalent to those used in the examples.

Polyclonal Antibodies

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats. Briefly, ROR1 antigen is utilizedto immunize the animal through intraperitoneal, intramuscular,intraocular, or subcutaneous injections, with an adjuvant such asFreund's complete or incomplete adjuvant. Following several boosterimmunizations, samples of serum are collected and tested for reactivityto ROR1. Particularly preferred polyclonal antisera will give a signalon one of these assays that is at least three times greater thanbackground. Once the titer of the animal has reached a plateau in termsof its reactivity to ROR1, larger quantities of antisera may be readilyobtained either by weekly bleedings, or by exsanguinating the animal.

Monoclonal Antibodies

Monoclonal antibody (mAb) technology can be used to obtain mAbs to ROR1.Briefly, hybridomas are produced using spleen cells from mice immunizedwith human ROR1 antigens. The spleen cells of each immunized mouse arefused with mouse myeloma Sp 2/0 cells, for example using thepolyethylene glycol fusion method of Galfre, G. and Milstein, C.,Methods Enzymol., 73:3-46 (1981). Growth of hybridomas, selection in HATmedium, cloning and screening of clones against antigens are carried outusing standard methodology (Galfre, G. and Milstein, C., MethodsEnzymol., 73:3-46 (1981)).

HAT-selected clones are injected into mice to produce large quantitiesof mAb in ascites as described by Galfre, G. and Milstein, C., MethodsEnzymol., 73:3-46 (1981), which can be purified using protein A columnchromatography (BioRad, Hercules, Calif.). mAbs are selected on thebasis of their (a) specificity for ROR-1, (b) high binding affinity, (c)isotype, and (d) stability.

mAbs can be screened or tested for ROR1 specificity using any of avariety of standard techniques, including Western Blotting (Koren, E. etal., Biochim. Biophys. Acta 876:91-100 (1986)) and enzyme-linkedimmunosorbent assay (ELISA) (Koren, E. et al., Biochim. Biophys. Acta876:91-100 (1986)).

Humanized Antibodies

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA86:10029-10033, 1989 and WO 90/07861, each incorporated by reference).Human antibodies can be obtained using phage-display methods (see, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047). In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity may be selected by affinity enrichment.

Antibody Fragments

It may be desirable to produce and use functional fragments of a mAb fora particular application. The well-known basic structure of a typicalIgG molecule is a symmetrical tetrameric Y-shaped molecule ofapproximately 150,000 to 200,000 daltons consisting of two identicallight polypeptide chains (containing about 220 amino acids) and twoidentical heavy polypeptide chains (containing about 440 amino acids).Heavy chains are linked to one another through at least one disulfidebond. Each light chain is linked to a contiguous heavy chain by adisulfide linkage. An antigen-binding site or domain is located in eacharm of the Y-shaped antibody molecule and is formed between the aminoterminal regions of each pair of disulfide linked light and heavychains. These amino terminal regions of the light and heavy chainsconsist of approximately their first 110 amino terminal amino acids andare known as the variable regions of the light and heavy chains.

In addition, within the variable regions of the light and heavy chainsthere are hypervariable regions that contain stretches of amino acidsequences, known as complementarity determining regions (CDRs). CDRs areresponsible for the antibody's specificity for one particular site on anantigen molecule called an epitope. Thus, the typical IgG molecule isdivalent in that it can bind two antigen molecules because eachantigen-binding site is able to bind the specific epitope of eachantigen molecule. The carboxy terminal regions of light and heavy chainsare similar or identical to those of other antibody molecules and arecalled constant regions. The amino acid sequence of the constant regionof the heavy chains of a particular antibody defines what class ofantibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes ofantibodies contain two or more identical antibodies associated with eachother in multivalent antigen-binding arrangements.

Fab and F(ab′)₂ fragments of mAbs that bind ROR-1 can be used in placeof whole mAbs. Because Fab and F(ab′)₂ fragments are smaller than intactantibody molecules, more antigen-binding domains are available than whenwhole antibody molecules are used. Proteolytic cleavage of a typical IgGmolecule with papain is known to produce two separate antigen bindingfragments called Fab fragments which contain an intact light chainlinked to an amino terminal portion of the contiguous heavy chain viadisulfide linkage. The remaining portion of the papain-digestedimmunoglobin molecule is known as the Fc fragment and consists of thecarboxy terminal portions of the antibody left intact and linkedtogether via disulfide bonds. If an antibody is digested with pepsin, afragment known as an F(ab′)₂ fragment is produced which lacks the Fcregion but contains both antigen-binding domains held together bydisulfide bonds between contiguous light and heavy chains (as Fabfragments) and also disulfide linkages between the remaining portions ofthe contiguous heavy chains (Handbook of Experimental Immunology. Vol 1:Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications,Oxford (1986)).

Recombinant DNA methods have been developed which permit the productionand selection of recombinant immunoglobulin peptides which are singlechain antigen-binding polypeptides known as single chain Fv fragments(ScFvs or ScFv antibodies). Further, ScFvs can be dimerized to produce adiabody. ScFvs bind a specific epitope of interest and can be producedusing any of a variety of recombinant bacterial phage-based methods, forexample as described in Lowman et al. (1991) Biochemistry, 30,10832-10838; Clackson et al. (1991) Nature 352, 624-628; and Cwirla etal. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382. These methods areusually based on producing genetically altered filamentous phage, suchas recombinant M13 or fd phages, which display on the surface of thephage particle a recombinant fusion protein containing theantigen-binding ScFv antibody as the amino terminal region of the fusionprotein and the minor phage coat protein g3p as the carboxy terminalregion of the fusion protein. Such recombinant phages can be readilygrown and isolated using well-known phage methods. Furthermore, theintact phage particles can usually be screened directly for the presence(display) of an antigen-binding ScFv on their surface without thenecessity of isolating the ScFv away from the phage particle.

To produce an ScFv, standard reverse transcriptase protocols are used tofirst produce cDNA from mRNA isolated from a hybridoma that produces amAb for targeting the ROR1 antigen. The cDNA molecules encoding thevariable regions of the heavy and light chains of the mAb can then beamplified by standard polymerase chain reaction (PCR) methodology usinga set of primers for mouse immunoglobulin heavy and light variableregions (Clackson (1991) Nature, 352, 624-628). The amplified cDNAsencoding mAb heavy and light chain variable regions are then linkedtogether with a linker oligonucleotide in order to generate arecombinant ScFv DNA molecule. The ScFv DNA is ligated into afilamentous phage plasmid designed to fuse the amplified cDNA sequencesinto the 5′ region of the phage gene encoding the minor coat proteincalled g3p. Escherichia coli bacterial cells are than transformed withthe recombinant phage plasmids, and filamentous phage grown andharvested. The desired recombinant phages display antigen-bindingdomains fused to the amino terminal region of the minor coat protein.Such “display phages” can then be passed over immobilized antigen, forexample, using the method known as “panning”, see Parmley and Smith(1989) Adv. Exp. Med. Biol. 251, 215-218; Cwirla et al. (1990) Proc.Natl. Acad. Sci. USA 87, 6378-6382, to adsorb those phage particlescontaining ScFv antibody proteins that are capable of binding antigen.The antigen-binding phage particles can then be amplified by standardphage infection methods, and the amplified recombinant phage populationagain selected for antigen-binding ability. Such successive rounds ofselection for antigen-binding ability, followed by amplification, selectfor enhanced antigen-binding ability in the ScFvs displayed onrecombinant phages. Selection for increased antigen-binding affinity maybe made by adjusting the conditions under which binding takes place torequire a tighter binding activity.

Another method to select for enhanced antigen-binding activity is toalter nucleotide sequences within the cDNA encoding the binding domainof the ScFv and subject recombinant phage populations to successiverounds of selection for antigen-binding activity and amplification (seeLowman et al. (1991) Biochemistry 30, 10832-10838; and Cwirla et al.(1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382).

Once an ScFv is selected, the recombinant ROR1 antibody can be producedin a free form using an appropriate vector in conjunction with E. colistrain HB2151. These bacteria actually secrete ScFv in a soluble form,free of phage components (Hoogenboom et al. (1991) Nucl. Acids Res. 19,4133-4137). The purification of soluble ScFv from the HB2151 bacteriaculture medium can be accomplished by affinity chromatography usingantigen molecules immobilized on a solid support such as AFFIGEL™(BioRad, Hercules, Calif.).

Other developments in the recombinant antibody technology demonstratepossibilities for further improvements such as increased avidity ofbinding by polymerization of ScFvs into dimers and tetramers (seeHolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6444-6448).

Because ScFvs are even smaller molecules than Fab or F(ab′)₂ fragments,they can be used to attain even higher densities of antigen bindingsites per unit of surface area when immobilized on a solid supportmaterial than possible using whole antibodies, F(ab′)₂, or Fabfragments. Furthermore, recombinant antibody technology offers a morestable genetic source of antibodies, as compared with hybridomas.Recombinant antibodies can also be produced more quickly andeconomically using standard bacterial phage production methods.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′).sub.2, Fd, Fvs, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv), fragmentscomprising either a VL or VH domain, fragments produced by a Fabexpression library, and anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to ROR1 antibodies disclosed herein). ScFvmolecules are known in the art and are described, e.g., in U.S. Pat. No.5,892,019. Immunoglobulin or antibody molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Examples of scFv to human ROR1 include SEQ ID NO:21, SEQ ID NO:22, SEQID NO:23 and SEQ ID NO:24.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are antigen-binding fragments also comprisingany combination of variable region(s) with a hinge region, CH1, CH2, andCH3 domains. Antibodies or immunospecific fragments thereof of thepresent invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al.

Recombinant Antibody Production

To produce antibodies described herein recombinantly, nucleic acidsencoding light and heavy chain variable regions, optionally linked toconstant regions, are inserted into expression vectors. The light andheavy chains can be cloned in the same or different expression vectors.For example, the heavy and light chains of SEQ ID NOs: 1-5 can be usedaccording to the present invention. The teachings of U.S. Pat. No.6,287,569 to Kipps et al., incorporated herein by reference in itsentirety, and the methods provided herein can readily be adapted bythose of skill in the art to create the vaccines of the presentinvention. The DNA segments encoding antibody chains are operably linkedto control sequences in the expression vector(s) that ensure theexpression of antibody chains. Such control sequences may include asignal sequence, a promoter, an enhancer, and a transcriptiontermination sequence.

Expression vectors are typically replicable in the host organisms eitheras episomes or as an integral part of the host chromosome. E. coli isone prokaryotic host particularly useful for expressing antibodies ofthe present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilus, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication) and regulatory sequences such as alactose promoter system, a tryptophan (trp) promoter system, abeta-lactamase promoter system, or a promoter system from phage lambda.Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Mammaliantissue cell culture can also be used to express and produce theantibodies of the present invention (see, e.g., Winnacker, From Genes toClones VCH Publishers, N.Y., 1987). Eukaryotic cells are preferred,because a number of suitable host cell lines capable of secreting intactantibodies have been developed. Preferred suitable host cells forexpressing nucleic acids encoding the immunoglobulins of the inventioninclude: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line; baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary-cells (CHO); mouse sertoli cells; monkeykidney cells (CV1 ATCC CCL 70); african green monkey kidney cells(VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); and TRI cells.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell. Calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation can be used for other cellularhosts (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 2nd ed., 1989). When heavy and lightchains are cloned on separate expression vectors, the vectors areco-transfected to obtain expression and assembly of intactimmunoglobulins. After introduction of recombinant DNA, cell linesexpressing immunoglobulin products are cell selected. Cell lines capableof stable expression are preferred (i.e., undiminished levels ofexpression after fifty passages of the cell line).

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see, e.g., Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred.

Multiple Specific Antibodies, Antibody Immunoconjugates and FusionMolecules

ROR1 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may be “multispecific,” e.g., bispecific,trispecific or of greater multispecificity, meaning that it recognizesand binds to two or more different epitopes present on one or moredifferent antigens (e.g., proteins) at the same time. Thus, whether anROR1 antibody is “monospecific” or “multispecific,” e.g., “bispecific,”refers to the number of different epitopes with which a bindingpolypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an ROR1antibody, binding polypeptide or antibody. Each binding domainspecifically binds one epitope. When an ROR1 antibody, bindingpolypeptide or antibody comprises more than one binding domain, eachbinding domain may specifically bind the same epitope, for an antibodywith two binding domains, termed “bivalent monospecific,” or todifferent epitopes, for an antibody with two binding domains, termed“bivalent bispecific.” An antibody may also be bispecific and bivalentfor each specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent antibodies, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tuft et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

The present invention includes multispecific ROR1 antibodies. Forexample, a bispecific antibody comprised of two scFv antibody fragments,both of which bind ROR1. The scFv antibody fragments may bind the sameor different epitopes on ROR1. As an additional example, themultispecific antibody may be a diabody which binds to the epitopes ofthe antibodies with a heavy chain variable region selected from thegroup consisting of SEQ ID NO:1. SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:39 or SEQ ID NO:42 and a light chainvariable region selected from the group consisting of SEQ ID NO:3. SEQID NO:7, SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO:19, SEQ ID NO:41 or SEQID NO:45.

The invention further extends to fusion proteins. Fusion proteins arechimeric molecules that comprise, for example, an immunoglobulinantigen-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

ROR1 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, ROR1-specificantibodies may be recombinantly fused or conjugated to molecules usefulas labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387. Radiolabled ROR1 antibodies of the inventionwill be particularly useful, while antibody drug conjugates (ADCs)remain to be developed.

ROR1 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention include derivatives that are modified, i.e., bythe covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody binding ROR1. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

ROR1 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. ROR1-specific antibodies may be modified by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the ROR1-specific antibody,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini, or on moieties such as carbohydrates. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given ROR1-specificantibody.

The present invention also provides for fusion proteins comprising anROR1 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused may be useful for function or is useful totarget the ROR1 polypeptide expressing cells. In one embodiment, afusion protein of the invention comprises a polypeptide having the aminoacid sequence of any one or more of the VH regions of an antibody of theinvention or the amino acid sequence of any one or more of the VLregions of an antibody of the invention or fragments or variantsthereof, and a heterologous polypeptide sequence.

In another embodiment, a fusion protein for use in the treatment methodsdisclosed herein comprises a polypeptide having the amino acid sequenceof any one, two, three of the VH-CDRs selected from the group consistingof SEQ ID NO:27, SEQ ID NO: 28 and SEQ ID NO:29 of an ROR1-specificantibody, or fragments, variants, or derivatives thereof, or the aminoacid sequence of any one, two, three of the VL-CDRs selected from thegroup consisting of SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32 of anROR1-specific antibody, or fragments, variants, or derivatives thereof,and a heterologous polypeptide sequence. In one embodiment, the fusionprotein comprises a polypeptide having the amino acid sequence of aVH-CDR3 of an ROR1-specific antibody of the present invention, orfragment, derivative, or variant thereof, and a heterologous polypeptidesequence, which fusion protein specifically binds to at least oneepitope of ROR1. In another embodiment, a fusion protein comprises apolypeptide having the amino acid sequence of at least one VH region ofa ROR1-specific antibody of the invention and the amino acid sequence ofat least one VL region of an ROR1-specific antibody of the invention orfragments, derivatives or variants thereof, and a heterologouspolypeptide sequence. Preferably, the VH and VL regions of the fusionprotein correspond to a single source antibody (or scFv or Fab fragment)that specifically binds at least one epitope of ROR1. In yet anotherembodiment, a fusion protein for use in the diagnostic and treatmentmethods disclosed herein comprises a polypeptide having the amino acidsequence of any one, two, three or more of the VH CDRs of anROR1-specific antibody and the amino acid sequence of any one, two,three or more of the VL CDRs of an ROR1-specific antibody, or fragmentsor variants thereof, and a heterologous polypeptide sequence.Preferably, two, three, four, five, six, or more of the VH-CDR(s) orVL-CDR(s) correspond to single source antibody (or scFv or Fab fragment)of the invention. Nucleic acid molecules encoding these fusion proteinsare also encompassed by the invention.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

The invention provides for a particularly preferred anti-human ROR1antibody; i.e., an isolated anti-ROR1 antibody having the same bindingspecificity as antibody 99961. In one aspect, the antibody binds to theIg-like domain that is contiguous with the CRD domain of ROR1. In anadditional aspect, the antibody binds to amino acids 42-160 of hROR1. Ina further aspect, the antibody binds to amino acids 130-160 of ROR-1. Inanother aspect, the antibody requires glutamic acid at position 138 ofhROR1 to be present for binding

In an additional embodiment, the invention provides for an isolatedanti-ROR1 antibody comprising a heavy chain variable region is selectedfrom the group consisting of SEQ ID. NO:1, SEQ ID. NO:5, SEQ ID. NO:9,SEQ ID. NO:13, and SEQ ID. NO:17, and the light chain variable region isselected from the group consisting of SEQ ID. NO:3, SEQ ID. NO:7, SEQID. NO:11, SEQ ID. NO:15 and SEQ ID. NO:19. In one aspect, the antibodyaccording the heavy chain variable region is SEQ ID NO:5 and the lightchain variable region is SEQ ID NO:7.

In one embodiment, the invention provides for an isolated anti-humanROR1 antibody comprising a heavy chain variable region comprised ofCDR1, CDR2 and CDR3 selected from the group consisting of SEQ ID. NO:27,SEQ ID. NO:28, SEQ ID. NO:29, SEQ ID. NO:33, SEQ ID NO:34 and SEQ ID.NO:35, and the light chain variable region comprised of CDR1, CDR2 andCDR3 selected from the group consisting of SEQ ID. NO:30, SEQ ID. NO:31,SEQ ID. NO:32, SEQ ID. NO:36, SEQ ID NO:37 and SEQ ID. NO:38. In oneaspect the a heavy chain variable region comprised of CDR1, CDR2 andCDR3 is comprised of SEQ ID. NO:27, SEQ ID. NO:28 and SEQ ID. NO:29, andthe light chain variable region comprised of CDR1, CDR2 and CDR3selected from the group consisting of SEQ ID. NO:30, SEQ ID. NO:31 andSEQ ID. NO:32.

In a further embodiment, the invention provides for an anti-human ROR1antibody with a binding affinity greater than 41 nM. In an aspect, theantibody binding affinity is between about 500 pM and about 6 nM. In oneaspect, the antibody binding affinity is about 800 pM.

In another aspect, the antibody inhibits metastasis. In an additionalaspect, the antibody internalizes and inhibits cell migration. In afurther aspect, the antibody internalizes and down modulates vimentin,snail1/2 or ZEB. In another aspect, the antibody is human, humanized orchimeric. In one aspect, the antibody is 99961, 99961.1, 99961.2,99961.3 or 99961.4. In a preferred aspect, the antibody is 99961.1.

One embodiment of the invention provides for a pharmaceuticalformulation comprising the antibody against ROR1 and a pharmaceuticallyacceptable carrier. In an additional embodiment, the invention providesan isolated nucleic acid encoding the antibody against ROR1. In anotherembodiment, the invention provides for an expression vector comprisingthe nucleic acid according to nucleic acid encoding an antibody againstROR1. In an additional embodiment, the invention provides for a hostcell comprising the nucleic acid encoding an antibody against ROR1. In afurther embodiment, the invention provides for a method of producing ananti-ROR1 antibody comprising culturing the host cells under conditionsto produce the antibody. In one aspect, the method of producing anantibody further comprises recovering the antibody.

As shown in the examples, anti-ROR1 antibody D10 inhibits mouse andhuman CLL engraftment, can direct complement-dependent cytotoxicity,induces significant reduction in leukemic burden, and blocks metastasisof breast cancer cells to lung and bone.

D10 has been shown to have biologic activity while other known anti-ROR1antibodies (e.g., 4A5 and K19) do not exhibit biologic activity despite4A5 having a significantly higher binding affinity for ROR1. Antibody4A5 has been shown to bind to different epitopes than D10. It has alsobeen shown that a subset of cancer patients, in which the cancer isROR+, antisera to ROR1 is produced. A further subset of patients makeantibodies that inhibit Wnt5a activity, thus leading to the conclusionthat not all ROR1 antibodies have biologic activity.

As described further in the Examples, epitope mapping was performed todetermine the epitope of D10 and 4A5. These studies determined that D10binds to an epitope at the C-terminus of the Ig like domain that iscontiguous to the CRD on ROR1. The epitope for 4A5 was also mapped tothe Ig like domain, but closer to the amino terminal of the domain.These findings have led to the conclusion that antibodies which bind tothe same epitope as D10 will inhibit ROR1 biologic activity whileantibodies that bind elsewhere may not.

As shown in the examples, high affinity antibodies, i.e. 99961, werederived using the D10 epitope to select for high affinity recombinantantibodies. One of the selected antibodies, 99961 has a significantlyhigher binding affinity for ROR1 than D10. The 99961 antibody has 50×greater binding affinity than D10, i.e. 800 pM v. 41 nM. Additionally,99961 was humanized generating four different antibodies. Experimentsconfirmed that 99961 has the same epitope as D10. Experiments confirmedthat this epitope is not expressed on normal hematopoietic stem andprogenitor cells. Further, 99961 does not cross react with normal adulttissues. This antibody also demonstrated activity against CLL cells,activity in ROR+ primary AML and induction of ROR1 internalization.

Vaccines

Additionally, the invention provides a vaccine for the treatment orprevention of cancer or the inhibition of metastasis in a subject thatconsists of a pharmaceutically acceptable composition of an isolated orsynthetically produced ROR1 binding peptide. The invention also providesfor a ROR1 binding peptide with at least 95% sequence identity to theROR-1 binding region of D10. In a further aspect, the invention providesfor a ROR1 binding peptide with at least 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the binding region of D10. In one aspect, thebinding region of D10 is VATNGKEVVSSTGVLFVKFGPC. In an additionalaspect, the binding region of D10 is EVVSSTGVLFVKFGPC. In one aspect theD10 binding region is at least 22 amino acids. In a further aspect, theD10 binding region is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21 or 22 amino acids.

The present invention also provides for use of ROR1 binding peptidevaccines against diseases, such as a lymphoma, e.g., CLL, that involvethe expression of ROR1. Because normal adult tissues do not appear toexpress ROR-1, it represents a tumor-specific antigen that can betargeted in active immune therapy. For example, the levels of ROR1 canbe down-regulated by administering to the patient a therapeuticallyeffective amount of a ROR1 binding peptide vaccine that produces inanimals a protective or therapeutic immune response against ROR1 and theeffects of its expression. The vaccines can include peptides. Methods ofusing such peptides include use in vaccines and for generatingantibodies against ROR1. The ROR1 binding peptide may also include animmune adjuvant. The immunoadjuvant may be an immunogenic carrier moietyconjugated to the binding peptide. In one aspect, the immunogeniccarrier moiety is a peptide. Examples of a suitable carrier for thevaccine further comprises an immunogenic adjuvant. In a further aspect,the adjuvant is an immunogenic carrier moeity conjugated to the bindingpeptide. The immunogenic carrier moiety may be a carrier peptide, suchas keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),ovalbumin, aluminum hydroxide or other pharmaceutically acceptableimmune adjuvant. Examples of pharmaceutically acceptable immuneadjuvants can be found in Methods in Molecular Medicine, Vol. 42:Vaccine adjuvants: Preparation, Methods and Research Protocols; Editedby D. T. O'Hagan; Humana Press Inc., Totowa N.J. and European Agency forthe Evaluation of Medicinal Products, Committee for ProprietaryMedicinal Products, Guidelines on Adjuvants in Vaccines, London 2004.Typically the vaccine composition will also include a pharmaceuticallyacceptable carrier or diluent.

In one embodiment the invention provides for a vaccine against ROR-1expressing cells, the vaccine comprising a pharmaceutically acceptablecomposition of an isolated or synthetically produced peptide having anamino acid sequence with at least 95% sequence identity to the ROR-1binding region of antibody D10. In one aspect, the vaccine the aminoacid sequence of the ROR-1 binding region of antibody D10 isVATNGKEVVSSTGVLFVKFGPC. In a further aspect, the vaccine the amino acidsequence of the ROR-1 binding region of antibody D10 isEVVSSTGVLFVKFGPC. In another aspect, the ROR1 expressing cell is acancer cell. In an additional aspect, the cancer cell is from a B cellleukemia, lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, or adrenal cancer.

In another embodiment, the invention provides for a vaccine comprising aROR1 binding peptide having an amino acid sequence with at least 95%sequence identity to VATNGKEVVSSTGVLFVKFGPC and a pharmaceuticallyacceptable carrier. In one aspect, the peptide is mammalian. In anadditional aspect, the peptide is chimeric and/or of human, mouse, rat,porcine, bovine, primate, feline, canine, rabbit, goat, chicken orursine origin. In another aspect, the vaccine further comprises animmunogenic adjuvant. In a further aspect, the adjuvant is animmunogenic carrier peptide conjugated to the binding peptide. In oneaspect, the amino acid sequence of the binding peptide isVATNGKEVVSSTGVLFVKFGPC. In another aspect, the immunogenic carrierpeptide is keyhole limpet hemocyanin (KLH). The vaccine furthercomprises an immunogenic adjuvant. In a further aspect, the adjuvant isan immunogenic carrier moiety conjugated to the binding peptide. In oneaspect, the amino acid sequence of the binding peptide isVATNGKEVVSSTGVLFVKFGPC. The immunogenic carrier moiety may be a carrierpeptide, such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA), ovalbumin, aluminum hydroxide or other pharmaceuticallyacceptable immune adjuvant. Examples of pharmaceutically acceptableimmune adjuvants can be found in Methods in Molecular Medicine, Vol. 42:Vaccine adjuvants: Preparation, Methods and Research Protocols; Editedby D. T. O'Hagan; Humana Press Inc., Totowa N.J. and European Agency forthe Evaluation of Medicinal Products, Committee for ProprietaryMedicinal Products, Guidelines on Adjuvants in Vaccines, London 2004.

In another embodiment, the invention provides for a vaccine comprising aROR1 binding peptide having an amino acid sequence with at least 95%sequence identity to EVVSSTGVLFVKFGPC and a pharmaceutically acceptablecarrier. In one aspect, the peptide is mammalian. In an additionalaspect, the peptide is chimeric and/or of human, mouse, rat, porcine,bovine, primate, feline, canine, rabbit, goat, chicken or ursine origin.In another aspect, the vaccine further comprises an immunogenicadjuvant. In a further aspect, the adjuvant is an immunogenic carrierpeptide conjugated to the binding peptide. In one aspect, the amino acidsequence of the binding peptide is EVVSSTGVLFVKFGPC. The immunogeniccarrier moiety may be a carrier peptide, such as keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) ovalbumin, aluminumhydroxide or other pharmaceutically acceptable immune adjuvant. Examplesof pharmaceutically acceptable immune adjuvants can be found in Methodsin Molecular Medicine, Vol. 42: Vaccine adjuvants: Preparation, Methodsand Research Protocols; Edited by D. T. O'Hagan; Humana Press Inc.,Totowa N.J. and European Agency for the Evaluation of MedicinalProducts, Committee for Proprietary Medicinal Products, Guidelines onAdjuvants in Vaccines, London 2004.

In an additional embodiment, the invention provides for a pharmaceuticalformulation comprising the vaccine comprising a ROR1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC and a pharmaceutically acceptable carrier.

In an additional embodiment, the invention provides for a pharmaceuticalformulation comprising the vaccine comprising a ROR1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC and a pharmaceutically acceptable carrier.

As shown in the examples, peptide vaccines were developed as shown inFIG. 18. Three peptides were used based on the epitopes of ROR1antibodies D10, 4A5 and K19. Animals were immunized with the threepeptides. All three peptides induced the production of ROR1 antisera.The results demonstrate that immunization with R22 peptide produced thegreatest antibody titer. As indicated in the examples, the ROR1 antiserabinds to ROR1, decreases leukemic burden, induce ROR1 internalization,mediate complement dependent cytotoxicity, inhibit breast cancer cellmigration and inhibit engraftment of ROR+ leukemia cells. Thus, theinvention provides a method to immunize patients against ROR1 to allowfor the induction of antibodies to inhibit the capacity of ROR+ cancercells to migrate and metastasize.

ROR1 Binding Peptide

In one embodiment, the invention provides for a ROR1 binding peptidecomprising the amino acid sequence selected from the group consistingof: SEQ ID NO:25 and SEQ ID NO:26. In one aspect, the peptide has anamino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC. In another aspect, the peptide has an amino acidpeptide sequence at least 95% sequence identity to EVVSSTGVLFVKFGPC. Inanother aspect, the binding peptide is mammalian. In an additionalaspect, the binding peptide is chimeric and/or of human, mouse, rat,porcine, bovine, primate, feline, canine, rabbit, goat, chicken orursine origin.

In an embodiment, the invention provides for a pharmaceuticalformulation comprising a ROR1 binding peptide comprising the amino acidsequence selected from the group consisting of: SEQ ID NO:25 and SEQ IDNO:26 and a pharmaceutically acceptable carrier.

In another embodiment, the invention provides for an isolated nucleicacid encoding a ROR1 binding peptide comprising the amino acid sequenceof SEQ ID NO:25 and SEQ ID NO:26. In another embodiment, the inventionprovides for an expression vector comprising the nucleic encoding a ROR1binding peptide comprising the amino acid sequence of SEQ ID NO:25 andSEQ ID NO:26. In a further embodiment, the invention provides for a hostcell comprising the nucleic acid encoding a ROR1 binding peptidecomprising the amino acid sequence of SEQ ID NO:25 and SEQ ID NO:26. Inan additional embodiment, the invention provides for a method ofproducing a peptide comprising culturing the host cell encoding a ROR1binding peptide comprising the amino acid sequence of SEQ ID NO:25 andSEQ ID NO:26 under conditions to produce the binding peptide. In oneaspect, the method to produce a peptide further comprises recovering thebinding peptide.

Suppression of Metastasis

In one embodiment, the invention provides for a method of suppressingmetastasis of ROR-1 expressing cancer, the method comprising disruptingepithelial-mesenchymal transition of tumor cells by administering anantibody having the binding specificity of monoclonal antibody 99961, avaccine comprised of a peptide having an amino acid sequence with atleast 95% sequence identity to the ROR-1 binding region of antibody D10,a ROR-1 binding peptide having an amino acid sequence with at least 95%sequence identity to VATNGKEVVSSTGVLFVKFGPC or a ROR-1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC. In one aspect, the ROR-1 expressing cancer is B cellleukemia, lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, or adrenal cancer.

The examples provide evidence that ROR1 antibodies, binding peptides andvaccines have the ability to inhibit ROR+ cancer cells from migrating ormetastasizing.

Treatment

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

As used herein, the term “cancer” or “cancer cell” or “ROR1 expressingcancer” or “ROR1 expressing cancer cell” refers to all neoplastic cellgrowth and proliferation, whether malignant or benign, including alltransformed cells and tissues and all cancerous cells and tissues.Cancer includes, but is not limited to neoplasms, whether benign ormalignant, located in the: prostate, colon, abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract. Suchneoplasms, in certain embodiments, express, over-express, or abnormallyexpress ROR1.

Cancer also includes but is not limited to B cell leukemia, lymphoma,CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin, pancreatic,testicular, bladder, uterine, prostate, and adrenal cancer.

The anti-ROR1 antibodies, ROR1 binding peptides and ROR1 vaccinesdescribed herein can be used for the treatment or prevention of a ROR1cancer or to inhibit metastasis of a ROR1 cancer cell in a subject.

Antibodies

In certain therapeutic embodiments, the selected antibody will typicallybe an anti-ROR1 antibody, which may be administered alone, or incombination with, or conjugated to, one or more combinatorialtherapeutic agents. When the antibodies described herein areadministered alone as therapeutic agents, they may exert a beneficialeffect in the subject by a variety of mechanisms. In certainembodiments, monoclonal antibodies that specifically bind hROR-1 arepurified and administered to a patient to neutralize one or more formsof hROR-1, to block one or more activities of hROR-1, or to block orinhibit an interaction of one or more forms of hROR-1 with anotherbiomolecule.

The immunotherapeutic reagents of the invention may include humanizedantibodies, and can be combined for therapeutic use with additionalactive or inert ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, e.g., immunogenic adjuvants, andoptionally with adjunctive or combinatorially active agents such asanti-neoplastic drugs.

In other embodiments, therapeutic antibodies described herein arecoordinately administered with, co-formulated with, or coupled to (e.g.,covalently bonded) a combinatorial therapeutic agent, for example aradionuclide, a differentiation inducer, a drug, or a toxin. Variousknown radionuclides can be employed, including ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I,¹⁸⁶Re, ¹⁸⁸Re, and ²¹¹At. Useful drugs for use in such combinatorialtreatment formulations and methods include methotrexate, and pyrimidineand purine analogs. Suitable differentiation inducers include phorbolesters and butyric acid. Suitable toxins include ricin, abrin, diptheriatoxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein. These combinatorial therapeutic agents canbe coupled to an anti-ROR1 antibody either directly or indirectly (e.g.,via a linker group). A direct reaction between an agent and an antibodyis possible when each possesses a substituent capable of reacting withthe other. For example, a nucleophilic group, such as an amino orsulfhydryl group, on one may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other. Alternatively, it may be desirable to couple a combinatorialtherapeutic agent and an antibody via a linker group as a spacer todistance an antibody from the combinatorial therapeutic agent in orderto avoid interference with binding capabilities. A linker group can alsoserve to increase the chemical reactivity of a substituent on an agentor an antibody, and thus increase the coupling efficiency. It will befurther evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as a linker group.Coupling may be affected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues.

It may also be desirable to couple more than one agent to an anti-ROR1antibody. In one embodiment, multiple molecules of an agent are coupledto one antibody molecule. In another embodiment, more than one type ofagent may be coupled to one antibody. Regardless of the particularembodiment, immunoconjugates with more than one agent may be prepared ina variety of ways. For example, more than one agent may be coupleddirectly to an antibody molecule, or linkers which provide multiplesites for attachment can be used. Alternatively, a carrier can be used.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration is intravenous,intramuscular, or subcutaneous.

It will be evident that the precise dose of the antibody/immunoconjugatewill vary depending upon such factors as the antibody used, the antigendensity, and the rate of clearance of the antibody. A safe and effectiveamount of an anti-ROR1 agent is, for example, that amount that wouldcause the desired therapeutic effect in a patient while minimizingundesired side effects. Generally, a therapeutically effective amount isthat sufficient to promote production of one or more cytokines and/or tocause complement-mediated or antibody-dependent cellular cytotoxicity.The dosage regimen will be determined by skilled clinicians, based onfactors such as the exact nature of the condition being treated, theseverity of the condition, the age and general physical condition of thepatient, and so on.

In an additional embodiment, the invention provides a method fortreating or preventing a cancer in a subject, the method comprisingadministering to the subject an antibody having the binding specificityof monoclonal antibody 99961, a vaccine comprised of a peptide having anamino acid sequence with at least 95% sequence identity to the humanROR-1 binding region of antibody D10, a ROR-1 binding peptide having anamino acid sequence with at least 95% sequence identity toVATNGKEVVSSTGVLFVKFGPC or a ROR-1 binding peptide having an amino acidsequence with at least 95% sequence identity to EVVSSTGVLFVKFGPC. In oneaspect, the cancer is B cell leukemia, lymphoma, CLL, AML, B-ALL, T-ALL,ovarian, colon, lung, skin, pancreatic, testicular, bladder, uterine,prostate, or adrenal cancer.

Inhibition of metastasis by targeting ROR1.

The spread of neoplastic cells from its original site to distant areasof the body is responsible for 90% of cancer-related deaths. Themetastatic process includes the physical translocation of primary tumorcells to a distant organ and subsequent colonization. Somepoor-prognostic gene signatures suggest that cells in some primarytumors are predisposed to metastasis. However, understanding of themolecular and cellular determinants of metastasis is limited, and theprocesses whereby tumor cells undergo this event are largely unknown.Recent attention has focused on a cell-biological program called theepithelial-mesenchymal transition (EMT), which now is considered tofactor prominently in tumor progression, acquisition of motility,invasiveness, metastasis, and self-renewal traits.

EMT confers on neoplastic epithelial cells the biological traits neededto accomplish most of the steps of the invasion-metastasis cascade. Inboth normal development and cancer metastasis, EMT appears regulated bycontextual signals that epithelial cells receive from theirmicroenvironment. Through use of multiple pathways involved in embryonicmorphogenesis and wound healing, cancer cells can concomitantly acquireattributes that enable invasion and metastasis.

Work to define cancer stem cells (CSCs) that can account for metastasisor relapse of cancer after therapy has identified a variety of traitsassociated with one or more subpopulations of CSCs within varioustumors. Some of these studies have found acquisition of phenotypiccharacteristic of cells in EMT can induce non-CSCs to enter into aCSC-like state. Therefore, metastatic cancer cells, which havepresumably undergone EMT, may exhibit a CSC phenotype and acquireinvasive properties that promote survival in the circulation,extravasation into a distant organ, angiogenesis, and uncontrolledgrowth at the metastatic sites.

As detailed further in the Examples, high-level expression of ROR1 incancer cells is associated with higher rates of relapsed and/ormetastatic disease. The effects of ROR1 expression and silencing inpatients with adenocarcinoma of the breast, described in Example 1,illustrates practice of the invention to inhibit metastasis. As shown,silencing ROR1 expression in metastatic-prone breast cancer cell linesreverses phenotypic features associated with EMT and impairs migration,invasion, and metastasis in vitro and in vivo. Further, the inventiveantibodies specific for ROR1 inhibit metastases of human breast cancercells xenografted into immune-deficient mice. These studies identify apreviously unknown pathway for breast cancer metastasis and validateROR1 as a promising target for cancer treatment. Low ROR1 expressionlevels were correlated with longer metastasis-free survival, and moreimportantly, therapeutic targeting of ROR1 with anti-ROR1 antibodies caninhibit breast cancer metastasis development.

Metastasis is the spread of cancer cells from their primary location toother parts of the body. Once cancer becomes metastatic, it cannot beeffectively treated by surgery or radiation therapy. Moreover, thepredominant cause of cancer patient′ mortality is metastasis. Receptortyrosine kinases (RTKs) are known to play crucial roles in many cellularprocesses, including differentiation, proliferation, migration,angiogenesis and survival. Although ROR2 has been found to facilitatemelanoma and prostate cancer cell metastasis, there is not a significantdifference in ROR2 expression between aggressive and non-aggressivebreast cancer cell lines. However, expression of ROR1 has a strongcorrelation with the aggressive breast cancer cell lines.

While the invention is not limited by theories as to its mechanism ofaction, it is notable that ROR1 activates genes that encode proteinsimplicated in breast cancer metastasis, such as Snail-1, Snail-2,TCF8/ZEB, CK-19, Vimentin, CXCR4. AKT was recently reported to beinvolved with functions of metastasis, including EMT, resistance toapoptosis and angiogenesis. As demonstrated in the Examples, ROR1up-regulated AKT activity and exposure of MDA-MB-231 cells to anti-ROR1antibody D10 reduced p-AKT activity. These data suggest that inhibitionof ROR1-regulated AKT activation may be one mechanism by which D10exerts its anti-tumor effect.

With respect to breast cancer metastasis in particular, using geneexpression signatures it was found that expression of ROR1 in primarybreast tumors is associated with breast cancer metastasis includingbone, lung, and brain metastasis. Among 582 cases that were analyzed,the relapse rate was 55% in the ROR1high group compared to 37% in theROR1 low group. Importantly, this relapse rate increased to 63% in ROR1the 75th-100th group. ROR1 expression is also strongly correlated withclinically aggressive breast cancer tumor markers, including ER-, PR-,and Her2-. Although there was no statistically significant differencebetween the groups based on the breast cancer T-stage, the percentage ofROR1 high patients increased from 51% to 77% in the Ti and T4 stages,respectively. Organ specific metastasis (breast cancer to lung or bone)was significantly inhibited by ROR1 knockdown according to theinvention. These data suggest that ROR1 may regulate certain lung andbone specific-genes, such as CXCR4.

Human chemokines are comprised of a superfamily of 48 ligands that bindto 19 different G protein-coupled chemokine receptors. It has beenhypothesized that metastatic tumor cells can ‘hijack’ chemokinereceptor-mediated cell migration highways. Breast cancer tumor cellsexpress selected chemokine receptors including CXCR4 Inhibition of theCXCL12-CXCR4 axis according to the invention can block the in vivometastasis of the cell line MDA-MB 231 to the lung. MDA-MB-231 cellssilenced for ROR1 had lower expression of CXCR4 than parental MDA-MB-231or MDA-MB-231 transfected with CTRL-shRNA.

Using gene expression analysis, it was found that the expression of ROR1was also associated with lung (FIG. 1B), bone (FIG. 1C), and brain (FIG.1D) metastasis. Based on a hazard ratio analysis, ROR1 was determined tobe an even better predictor of overall, bone, and lung metastasis thanER, PR and HER2. ROR1 can also be a metastasis-related predictor genebased on the overall relapse rate of breast cancer patients overallrelapse by ER and ROR1 status. Although there was some differencebetween ER+ and ER- cases in the early stage of metastasis-freesurvival, only ROR1 low and ROR1 high cases had significantlydifferences in the late stages of metastasis-free survival. Thus, theinvention provides a path to inhibit metastasis and improve patientsurvival.

In general, the dosage of administered ROR1 antibodies, ROR1 antibodycomponents, binding peptide vaccine compositions, immunoconjugatesthereof and fusion proteins will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of antibody component, vaccine, immunoconjugateor fusion protein that is in the range of from about 1 ng/kg to 20 mg/kg(amount of agent/body weight of patient), although a lower or higherdosage also may be administered as circumstances dictate.

Administration of antibodies, antibody components, vaccines,immunoconjugates or fusion proteins to a patient can be intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, by perfusion through a regional catheter, orby direct intralesional injection. When administering therapeuticproteins, peptides or conjugates by injection, the administration may beby continuous infusion or by single or multiple boluses.

Those of skill in the art are aware that intravenous injection providesa useful mode of administration due to the thoroughness of thecirculation in rapidly distributing antibodies. Intravenousadministration, however, is subject to limitation by a vascular barriercomprising endothelial cells of the vasculature and the subendothelialmatrix. Still, the vascular barrier is a more notable problem for theuptake of therapeutic antibodies by solid tumors. Lymphomas haverelatively high blood flow rates, contributing to effective antibodydelivery. Intralymphatic routes of administration, such as subcutaneousor intramuscular injection, or by catherization of lymphatic vessels,also provide a useful means of treating lymphomas.

Preferably, ROR1 antibodies, binding peptides, immunoconjugates thereofand fusion proteins are administered at low protein doses, such as 20 to3000 milligrams protein per dose, given once, or repeatedly,parenterally. Alternatively, administration is in doses of 100 to 300milligrams protein per dose, or 300 to 1000 milligrams protein per dose,1000 to 2000 milligrams protein per dose.

The present invention also contemplates therapeutic methods in whichROR1 antibody components are radiolabeled or supplemented withradiolabeled immunoconjugate or fusion protein administration. In onevariation, ROR1 antibodies are administered as or with low-doseradiolabeled ROR1 antibodies or fragments. As an alternative, ROR1antibodies may be administered with low-dose radiolabeled ROR1-cytokineimmunoconjugates. Those of ordinary skill in the art will be familiarwith pharmaceutically acceptable radiolabelling molecules and theirappropriate dosing levels. For reference, consider “low doses” of¹³¹I-labeled immunoconjugates, wherein a preferable dosage is in therange of 15 to 40 mCi, while the most preferable range is 20 to 30 mCi.In contrast, a preferred dosage of ⁹⁰Y-labeled immunoconjugates is inthe range from 10 to 30 mCi, while the most preferable range is 10 to 20mCi.

The invention in all its aspects is illustrated further in the followingExamples. The Examples do not, however, limit the scope of theinvention, which is defined by the appended claims.

Example 1 ROR1 is Associated with Early Metastatic Relapse in BreastAdenocarcinoma

The transcriptome data in the GEO database on breast cancer cellsisolated from patients in a combined cohort of 582 patients wasinterrogated. Approximately two-thirds (426 of 582) of these cases didnot have detectable cancer in the regional lymph nodes at the time ofsurgery and were not administered adjuvant therapy. The remaining caseshad detectable disease in regional lymph nodes and received adjuvanthormonal therapy and/or chemotherapy. Among the 582 cases, 46% relapsed(n=270), and had a median metastasis-free survival time of 22.1 months.We segregated patients into three groups based upon their relativecancer-cell expression of ROR1. Patients with tumors having theupper-third level of ROR1 mRNA expression (ROR1_(H)) had a significantlyshorter metastasis-free survival than patients with tumors that had thelower-third-level (ROR1_(L)) or intermediate-level (ROR1_(M)) expressionof ROR1 (p<0.0001; FIG. 1A). Metastasis-free survival by organ sites wasexamined. It was found that patients with ROR1H tumors had higher ratesof metastasis to the lung (p=0.002; FIG. 45A), bone (p=0.004; FIG. 45B),or brain (p=0.04; FIG. 45C) than did patients with ROR1_(L) or ROR1_(M)breast cancers. ROR1_(H) cancers had significantly lower proportions oftumors with favorable prognostic features, such as estrogen/progesteronereceptors or HER2, than cancers with ROR1_(L) or ROR1_(M).

High-level expression of ROR1 also performed as an independent factor inpredicting shorter metastasis-free survival. Patients with ROR1_(H)tumors had a higher rate of metastasis, earlier relapse, and poorersurvival than patients with ROR1_(L/M) tumors, irrespective of ER, PR,or HER2 status (FIG. 46). Furthermore, interrogation of the GSE2034,GSE2603, GSE5327, and GSE12276 array data for EMT gene signatures inbreast cancer revealed that ROR1_(L) tumors had significantly higherexpression levels of genes associated with epithelial cells, such asCDH1 (encoding E-cadherin), TJP1 (encoding Z01), and TJP3 (encodingZ03), but lower expression-levels of genes associated with mesenchymalcells, such as SNAI1 (encoding Snail-1), SNAI2 (encoding Snail-2), CDH2(encoding N-Cadherin) or VIM (encoding Vimentin), than ROR1_(H) tumors(FIG. 1B).

Example 2 ROR1+ Breast-Cancer Cell Lines

Fourteen distinct breast-cancer epithelial cell lines expression of ROR1were examined, including six basal-type breast cancer cell lines andeight luminal-type breast cancer cell lines. The level of expression ofROR1 was significantly greater in basal-type breast cancer cell linesrelative to that in luminal-type breast-cancer cell lines, whichgenerally did not express ROR1. Moreover, the relative expression-levelsof ROR1 correlated with aggressive tumor phenotypes, such as triplenegative ER^(Neg)PR^(Neg)HER2/Neu^(Neg), and high-level migration andinvasion capacity in vitro.

ROR1 was silenced in highly-invasive, basal-type breast cancer celllines (e.g. MDA-MB-231) using short hairpin RNAs (shRNAs) that targetedeither of two different ROR1 sequences. Expression of ROR1 protein wasinhibited in cells transfected with either ROR1-shRNA1 or ROR1-shRNA2,in contrast to cells transfected with a control shRNA (CTRL-shRNA) (FIG.47A). Interrogation of the array data for gene-expression differencesbetween MDA-MB-231 transfected with CTRL-shRNA or ROR1-shRNA (GEOaccession: GSE31631) revealed that cells silenced for ROR1 had higherexpression-levels of KRT19 (encoding CK19), lower expression-levels ofCXCR4 and VIM than parental MDA-MB-231 or MDA-MB-231 transfected withCTRL-shRNA. These findings were confirmed by qRT-PCR (FIG. 47B, FIG.48A). Flow cytometry analyses also demonstrated that cell-surfaceexpression of CXCR4 was lower in cells silenced for ROR1 (FIG. 48B).

To assess the potential roles of ROR1 in the regulation of EMT, weexamined for EMT-associated markers in cells treated with CTRL-shRNA orROR1-shRNA. Suppressing expression of ROR1 with either ROR1-siRNA orROR1-shRNA1/2 in either of three distinct, basal-type breast-cancercell-lines (MDA-MB-231, HS-578T, or BT549) attenuated their expressionof mRNA and/or encoded proteins associated with EMT (e.g. vimentin,SNAIL-1/2, and ZEB1). Conversely, silencing ROR1 increased expression ofmRNA and encoded epithelial cytokeratins (e.g. CK-19). Although therewere no significant changes in the TJP1 mRNA encoding ZO-1 in any of the3 cell lines examined, cells silenced for ROR1 had higher expressionlevels of this tight junction protein, suggesting that ZO-1 might beunder post-transcriptional control (FIGS. 1C-D and FIG. 49). Finally,transfection of ROR1-negative MCF7 cells to express ROR1 decreasedexpression of epithelial proteins (e.g. E-Cadherin and CK19), andincreased expression of EMT transcriptional factors, such as SNAIL1/2(FIG. 1E).

In culture, MDA-MB-231, HS-578T, or BT549 cells typically had exhibiteda stellate morphology, which is similar to that of mesenchymal cells invitro. However, following transfection with ROR1-shRNA these cellsassumed a more spherical morphology, which was similar to that ofepithelial cells (FIG. 2A). Transfection of these cells with CTRL-shRNAdid not induce such changes. Furthermore, immunofluorescence stainingrevealed that transfection with ROR1-shRNA induced MDA-MB-231 cells toexpress modest levels of E-cadherin and higher-levels of CK-19, butreduced expression of vimentin (FIG. 2B). Similar results also wereobserved for HS-578T or BT549 cells. On the other hand, compared tountreated cells or cells transfected with a control vector,ROR1-negative MCF7 cells developed a morphologic resemblance tomesenchymal cells and had decreased expression of epithelial markers(e.g. CK19 and E-Cadherin), and increased expression of mesenchymalmarkers, such as vimentin, when transfected to express ROR1.Furthermore, cells silenced for ROR1 had less migration/invasioncapacity compared to that of cells treated with CTRL-shRNA (FIGS. 2E andF). It was also found that chemotaxis toward CXCL12 was significantlyreduced in cells silenced for ROR1 (FIG. 48). Virtually identicalresults were obtained using cells silenced with either ROR1-shRNA1 orROR1-shRNA2. Collectively, these results indicate that expression ofROR1 may contribute to EMT and tumor metastasis.

Example 3 Silencing ROR1 Inhibits Orthotopic Lung Metastasis

Cell Culture

The breast cancer cell lines MDA-MB-231, HS-578T, BT549, MDA-MB-415,MDA-MB-435s, MDA-MB-436, MDA-MB-157, MDA-MB-134, MCF7, BT-474,MDA-MB-453, SKBR3, MDA-MB-330, and BT-483 were obtained from AmericanType Culture Collection (ATCC) and maintained as previously described(Neve et al. Cancer Cell, 10:515 (2006)).

ROR1-Knockdown

Knockdown of ROR1 was achieved by targeting the sequences 5′-TCC GGA TTGGAA TTC CCA TG-3′ (shRNA1), and 5′-CTT TAC TAG GAG ACG CCA ATA-3′(shRNA2) as previously described (Zhang, S. Et al., Cancer Cell, 16:67(2009)). A nonspecific shRNA control was created by targeting thesequences 5′-AGC GGA CTA AGT CCA TTG C-3′. Virapower™ lentiviralexpression systems (Invitrogen) were used to express the shRNA accordingto the manufacturer's instructions. The ROR1-shRNA1 and CTRL-shRNA1constructs also encoded red fluorescence protein (RFP). Oligonucleotidesfor the ROR1-shRNA1 and CTRL-shRNA1 constructs were synthesized(Integrated DNA Technologies) and inserted into the RFP-pLKO.1 vector.ROR1-shRNA2 and CTRL-shRNA2 constructs were purchased from OpenBiosystems (Rockford, Ill.). The viral particles for infection of breastcancer cells lines were obtained by transfection of the 293-FT packagingcell line, and collected from cell supernatants at 48 and 72 hrspost-transfection. Supernatants were filtered and centrifuged at43,000×g to concentrate the viral particles, which were used to infectsub-confluent cultures in the presence of 5 μg/ml polybrene overnight.

Twenty-four hours post-transfection, cells were selected with 2 μg/mlpuromycin. Knockdown cells were sorted by flow cytometry using ananti-ROR1 mAb (4A5). Sorted cells stably expressing shRNA1 or shRNA2were designated ROR1-shRNA1 or ROR1-shRNA2, respectively. Pooledpopulations of knockdown cells, obtained in the first 10 generationafter cell sorting without subcloning, were injected into rag-/-γ-/-mice for in vivo experiments. The efficiency of the knockdown of ROR1was confirmed by quantitative PCR with reverse transcription (qRT-PCR)Sybr green gene expression assays (Applied Biosystems), or westernimmunoblot analysis (anti-ROR1 antibody, S4102, Cell Signaling).β2-microglobulin and actin were used as endogenous controls for qRT-PCRand western blot, respectively.

Trans-Well Migration and Invasion Assays

Cancer cells were conditioned overnight in Dulbecco's modified Eagle'smedium supplemented with 0.2% fetal bovine serum (FBS) without growthfactors. The following day, cells were trypsinized and resuspended in0.2% FBS DMEM media without growth factors. Tumors cells were seeded ata density of 25,000 cells per well into trans-well inserts (3 μM poresize, BD Falcon) for migration assays or at a density of 50,000 cellsper well into matrigel-coated, growth-factor-reduced, invasion chambers(8 μM pore size, BD Biosciences). Wells were washed with phosphatebuffered saline (PBS) and fixed with 4% parafomaldehyde after 6 h formigration assays or after 22 h for invasion assays. The cells on theapical side of each insert were removed by scraping. Cells that hadmigrated to the basal side of the membrane were stained and visualizedwith a Nikon inverted microscope.

Analysis of mRNA and Protein Expression

Total RNA was purified using the RNeasy kit (Qiagen) and 2 μg of eachsample was used for generating cDNA using the high-capacity cDNAReverser transcprition kits (ABI). Each cDNAs was analyzed in triplicateusing an ABI 7500 Fast Real-Time PCR System (Applied Biosystem). Proteinexpression levels were assessed by immunoblot analysis with cell lysates(40-60 μg) in lysis buffer (20 mM HEPES (pH 7.9), 25% glycerol, 0.5 NNaCl, 1 mM EDTA, 1% NP-40, 0.5 mM dithiothreitol, and 0.1% deoxycholate)containing protease inhibitors (Roche) using anti-ROR1 (Cell Signaling)and anti-β-actin antibodies (Cell Signaling).

Flow Cytometry

Breast cancer cells were stained or pool sorted by flow cytometry. Cellswere washed and resuspended in 2% bovine serum albumin (BSA) (Sigma) inPBS solution and stained for ROR1 expression using an A1ex488-conjugatedantibody (clone 4A5 or clone D10) or an A1ex488-conjugated IgG2b orIgG2a isotype control according to the manufacturer's protocol. Flowcytometry data were collected using a FACSCalibur cytometer (BDBiosciences) and analyzed using FlowJo software.

Immunofluorescence and Immunohistochemistry Analysis

Mouse lungs were fixed with 4% paraformaldehyde and embedded in paraffinor frozen in OCT for histopathological examination. The tissue sections(5 μm thick) were prepared and stained with hematoxylin & eosin (H&E) orp-AKT (Ser473, D9E, Cell Signaling), p-Creb (Ser133, 87G3, CellSignaling), CK-19 (RCK108, Dako), or Vimentin (D21H3, Cell Signaling)primary antibodies. Images were collected using a Delta Visionmicroscope and processed with SPOT software.

Analysis of Metastasis

Female Rag-/-γ-/- mice were injected with: a pool of parental MDA-MB-231ROR1-shRNA1 cells (group 1), and control shRNA cells for parentalMDA-MB-231 (group 2). Cells were injected intravenously through thelateral tail vein in 100 μl PBS (5×10⁵ for groups 1-2; 2×10⁵ for groups3-4) or administered by intracardiac injection in 100 μl PBS (1×10⁵ forgroups 5-6). Non-invasive bioluminescence imaging was performed weeklyby IVIS 200 imaging systems. All mice that had not previously died orappeared sick were euthanized at 3-4 wks post-injection, and their lungswere removed and fixed in 10% formalin.

To study the effect of ROR1 on the in vivo metastasis of a mammary padxenograft, breast cancer tumors were induced in eight-week-old femaleRag-/-γ-/- mice by injecting 100 μl of a single-cell suspension (1×10⁶viable cells/mouse) subcutaneously into the second fat pad area of theright abdominal mammary gland. The tumor size was measured every 3 days.The tumors were removed when the tumor volumes reach 300 mm³. To studythe therapeutic effect of anti-ROR1 monoclonal antibodies in breastcancer metastasis, breast cancer tumors were induced in eight-week-oldfemale Rag-/-γ-/- mice through intravenous injection 100 μl of asingle-cell suspension (5×10⁵ cells/mouse). Mouse IgG or anti-ROR1 mAbswere injected intravenously weekly. Non-invasive bioluminescence imagingwas performed weekly. Five weeks after establishment of the xenograft,mice were sacrificed and lungs were removed and fixed in 10% formalin.

Oncomine Gene Expression Data Analysis

A microarray dataset of 582 patients from the Pubmed GEO database(GE02603, GSE5327, GSE2034 and GSE12276) was compiled. These datasetswere transformed by log 2 and each microarray was centered to the medianof all probes. For each patient, metastasis free survival was defined asthe time interval between the surgery and the diagnosis of metastasis.Relative levels of ROR1 mRNA expression in human tissues were determinedby Oncomine Cancer Microarray database analysis (www.oncomine.org) of apublished gene expression data set. The data were log-2-transformed,with the median set to zero and s.d. set to one.

Statistical analyses. Comparisons between Kaplan-Meier curves wereperformed using the log rank test. Data are presented as means±standarderror of the mean (SEM). An Unpaired Student's t test was used tocompare two group unless otherwise indicated. A p<0.05 was consideredstatistically significant.

The performance of ROR1 in predicting metastasis-free survival wasanalyzed by multivariate analyses with Cox proportional hazardregression models. The hazard ratio of each covariate and its 95%confidence interval are reported. P-values are calculated based on theNormal Distribution, assessing the probability for the null hypothesis(hazard ratio=1, i.e. no prognostic significance) to be true.

The metastatic potential of CTRL-shRNA-transfected was compared toROR1-shRNA-transfected MDA-MB-231 cells that were stably transfectedusing a luciferase/GFP-expression vector in an orthotopic model (FIG.3A). Injection of 2.5-10×10⁵ cells into the subcutaneous mammary fat-padof immune-deficient RAG-/-γc-/- mice generated primary tumors at thesite of injection that we could monitor via bioluminescence. We did notobserve significant differences in the progressive increases inbioluminescence of tumors that resulted from injection ofCTRL-shRNA-transfected versus ROR1-shRNA-transfected cells until 3 ormore weeks after the injection of at least 1×10⁶ cells, as noted inprior studies. To examine for differences in the rates of ‘spontaneous’cancer metastasis, the primary tumors resulting from injection of 1×10⁶cells were surgically removed when they reached a volume of 300 mm³(dotted line, FIG. 3B). Because of different growth rates, the mediannumber of days from cell-injection to surgical removal of the primarytumors was significantly greater for mice injected with cells silencedfor ROR1 (40±2.5 days) than for mice that received equal numbers ofCTRL-shRNA-transfected cells (31±0.5 days) (FIG. 3B). The extirpatedprimary tumors had similar volume, weight, and ex vivo bioluminescence(FIG. 3, C to E). Following the removal of the primary tumor wemonitored for metastatic disease via bioluminescence. Animals injectedwith CTRL-shRNA-transfected cells had significantly greaterbioluminescence in the lung or liver at the time of primary-tumorexcision than did the mice engrafted with cells silenced for ROR1 at thelater time when they had their primary tumors excised (FIGS. 3, E andF). Animals injected with cells silenced for ROR1 had less detectableincrease in lung bioluminescence relative to that of mice injected withCTRL-shRNA-transfected cells (FIG. 3G). The animals were sacrificed 21days after their primary tumors were excised to examine the ex vivobioluminescence, size, and histology of the lung (FIG. 3, H to J) andliver (FIG. 3, K and L). The extirpated lungs and livers of miceinjected with CTRL-shRNA-transfected cells had significantly greaterbioluminescence and weight than those of mice injected withROR1-silenced cells. Moreover, the lungs and livers of mice injectedwith CTRL-shRNA-transfected cells universally had extensive metastaticdisease, which was not observed in the tissues of mice injected withROR1-silenced cells (FIGS. 3, J and L).

Example 4

Silencing ROR1 Inhibits Experimental Lung And Bone Metastasis

The ROR1-shRNA or CTRL-shRNA transfected MDA-MB-231 cells wasadministered to 6-week-old Rag^(−/−)γ^(−/−)mice via intravenous (5×10⁵cells) or intracardiac (1×10⁵ cells) injection to evaluate fordifferences in metastatic potential of cells injected into either thevenous or arterial blood. All animals that receivedCTRL-shRNA-transfected cells into the lateral tail vein died within 32days of injection due to lung metastasis. Animals that had equal numbersof ROR1-shRNA-transfected cells injected into the tail vein survivedsignificantly longer (FIG. 4A). Animals injected withCTRL-shRNA-transfected cells had 19-fold or 60-fold greaterbioluminescence in the lungs at day 21 or day 28, respectively, thanmice injected with cells silenced for ROR1 (FIG. 4B). We also sacrificedanimals in another experiment at various times to examine the lungs formetastatic disease. Whereas nascent metastatic foci were readilydetected at 3 days after injection of CTRL-shRNA-transfected cells, few,if any, metastatic foci could be detected in the lungs of animalsinjected with ROR1-silenced cells, even at later time points (FIG.4C-E). Moreover the lungs extirpated from mice injected withCTRL-shRNA-transfected cells had significantly greater ex vivobioluminescence and median weight (3-fold and 6-fold on days 21 and 28,respectively) than the lungs of mice injected with ROR1-silenced cells(FIG. 4F-G, data not shown). The metastatic foci that developed inanimals injected with CTRL-shRNA-transfected cells also expressed higherlevels of phospho-AKT and phospho-CREB and had higher proportions ofproliferating cells than the few metastatic foci that we detected inmice injected with ROR1-silenced cells, which instead expressed higherlevels of CK-19 and lower levels of vimentin (FIG. 47).

We also examined for metastatic disease following injection of 1×10⁵cells into the left cardiac ventricle. All mice that receivedCTRL-shRNA-transfected cells died within 30 days of this injection,whereas animals injected with ROR1-silenced cells survived significantlylonger (FIG. 4H). Mice injected with CTRL-shRNA-transfected cellsdeveloped substantial femoral/pelvic-area bioluminescence, which was notdetected in mice injected with tumor cells silenced for ROR1 (FIGS. 4, Iand J). We sacrificed animals on day 21 and found the isolatedfemoral/pelvic bones of mice injected with CTRL-shRNA-transfected cellshad high bioluminescence (FIG. 4K) due to extensive marrow metastasis(FIG. 4L), which was not apparent in mice injected with cells silencedfor ROR1.

Recent studies have found that different tissue-sites impose differentrequirements for the establishment of metastases by circulating cancercells. Human breast cancer cell lines BoM1833 and LM2-4175 were selectedfrom MDA-MB-231 to have different tissue tropism. BoM-1833preferentially metastasizes to the bone and LM2-4175 preferentiallymetastasizes to the lung. We found that each of these cell linesretained expression of ROR1 (FIG. 5A). Transfection of each cell-linewith ROR1-shRNA2 silenced expression of ROR1 (FIGS. 5B and C), allowingus to examine the ROR1-dependency of organ-specific metastasis followingintravenous injection of 2×10⁵ LM2-4175 or intracardiac injection of1×10⁵ BoM-1833 into 6-week-old RAG-/-γc-/- mice. Mice injected withLM2-4175 silenced for ROR1 had a significantly lower median increase inlung bioluminescence and significantly longer median survival than didmice injected with CTRL-shRNA-transfected LM2-4175 (FIGS. 5D and E).Consistent with these observations, the lungs of mice isolated 21 daysafter the injection of ROR1-silenced LM2-4175 had significantly lowermedian weight, ex vivo bioluminescence, and fewer and smaller metastaticfoci than mice injected with CTRL-shRNA-transfected LM2-4175 (FIG. 5F toH). Similarly, mice injected with BoM-1833 silenced for ROR1 hadsignificantly lower increases in skeletal bioluminescence than did miceinjected with equal numbers of CTRL-shRNA-transfected BoM-1833 (FigureSI and J). Moreover, necropsy of animals sacrificed 21 days afterintracardiac injection revealed few, if any, detectable metastatic fociin the bone or liver. This was in marked contrast to the extensivemetastatic disease detected at each of these sites in animals injectedwith CTRL-shRNA-transfected BoM-1833 (FIGS. 5J and K).

Example 5 An Anti-ROR1 Antibody Inhibits Cancer Metastasis

Monoclonal antibodies (mAb) specific for the extracellular domain ofROR1 were generated and one, designated D10, could induce rapiddown-modulation of surface ROR1 at 37° C. (FIG. 5A). Treatment ofMDA-MB-231 with D10 caused ROR1 internalization, as assessed viaconfocal microscopy (FIG. 5B). This resulted in significant reduction ofsurface ROR1, as assessed via flow cytometry using a different mAbspecific for a distinct, non-cross-blocking epitope of ROR1 (FIG. 5C).Treatment of MDA-MB-231 with D10 also reduced expression of cytoplasmicvimentin (FIG. 5D), which was bound to ROR1 in co-immune-precipitationstudies (FIG. 5E). Treatment with D10 also significantly inhibited themigration and invasion capacity of MDA-MB-231 in vitro (FIGS. 5F and G).D10 also could inhibit the migration/invasion capacity of other ROR1+cancer cell-lines (e.g. HS-578T and BT549 (FIG. 9)).

D10 was assessed for inhibition of invasion and metastasis of MDA-MB-231injected into the tail vein of RAG-/-γc-/- mice. Following injection of5×10⁵ cells, the mice were given an intravenous injection of control IgGor D10 at 5 mg/kg and then sacrificed 3 days later. The ex vivobioluminescence of the lungs from animals given D10 was significantlylower than that of animals treated with control IgG (FIG. 5H). Moreover,the lungs of animals that received control IgG had multiple metastaticfoci, which were not detectable in mice treated with D10. In anotherexperiment, each mouse received an intravenous injection of 5×10⁵MDA-MB-231 and then given 3 weekly intravenous injections of control IgGor D10 at 5 mg/kg. Mice treated with D10 developed significantly lesspulmonary bioluminescence than mice given control IgG (FIGS. 5, I andJ). When sacrificed at day 35, the lungs of mice treated with D10 hadsignificantly lower weight (FIG. 5K) and fewer metastatic foci (FIG. 5L)than the lungs of animals given control IgG. Collectively, these dataindicate that D10 can inhibit metastasis in immune-deficient mice.

In conclusion, it is hereby demonstrated that ROR1 can mediate breastcancer metastasis and that therapeutic targeting of ROR1 can retardbreast cancer metastasis development. Although embryonic stem cellsexpress detectable ROR1 protein and the loss of ROR1 can enhance heartand skeletal abnormalities in ROR2-deficient mice, major adult tissuesrarely express ROR1 protein, except at low levels in the pancreas andadipose tissue, providing the antibodies and methods for their use ofthe invention with ROR1 cancer specificity

Example 6 ROR1 High Affinity Antibodies

Epitope studies were performed on ROR1 antibody D10, described above. Aseries of chimeric proteins with stretches of human and mouse ROR1 weregenerated to map the epitope(s) recognized by D10 that can down-modulateROR1, effect reduction in expression of vimentin, and inhibitcancer-cell migration in vitro (a good surrogate marker of the cancer'scapacity to form metastases). The only region of ROR1 that is involvedis the Ig-like domain that is on the amino terminus of ROR1. Eachconstruct contains a chimeric Ig-like domain and human CRD and Kringledomain (mouse portion is light, human portion is dark). Only the Ig-likedomains are shown here (FIG. 6). These constructs were expressed infree-style 293 cells. Culture media was used immunoblot and purifiedproteins were used for ELISA. Since the D10 mAb anti-ROR1 recognizedhuman ROR1, but not mouse ROR1, finding which of these constructs couldor could not bind could help map the epitope recognized by D10. Theresults indicate that antibody D10 binds to ROR1 at the C-terminus ofthe Ig like domain contiguous to the CRD domain (FIG. 7). FIG. 8 showsthe mapping of the epitope for ROR1 antibody 4A5. As indicated the 4A5epitope differs from the D10 epitope.

As described above, an anti-ROR1 antibody, i.e. D10, can inhibitpulmonary metastasis of MDA-MB-231 cell in vivo. The D10 monoclonalantibody facilitates ROR1 receptor internalization (FIG. 9 A,B). TheMDA-MB-231 cells were stained with iso-A1ex647, or D10-A1ex647 for 30min on ice. The stained cells were then separated into two fractions.One fraction was kept on ice for 1 h and the other fractions weretransferred to 37° C. for 15 min, 30 min. Twenty four hours anti-ROR1antibody D10 treatment decrease ROR1 surface expression in MDA-MB-231cells (FIG. 9C). ROR1 forms complex with vimentin in breast cancerMDA-MB-231 cells (FIG. 9D). D10 antibody treatment in vitro coulddecrease vimentin expression (FIG. 9E). Anti-ROR1 antibodies decreasebreast cancer migration in vitro. (FIG. 9F). The D10 monoclonal antibodyinhibits MDA-MB-231 breast cancer early-stage (day 2) lung metastasis(FIG. 9G). The D10 monoclonal antibody inhibits MDA-MB-231 breast cancerlung metastasis (FIG. 9H). Xenograft mice were intravenously (i.v.)injected with 200 mg anti-ROR1 antibody on day 1, and 100 mg anti-ROR1antibody on day 3, 7, 14, and 21. The normalized photo fluxes from thelung of MDA-MB-231 bearing mice are shown. Representative mice injectedwith 5E5 MDA-MB-231 cells are shown in the dorsal position (FIG. 9I).Anti-ROR1 antibody treatment reduced the lung weight ofMDA-MB-231-bearing mice (FIG. 9J). Representative pulmonary H&Ehistology from MDA-MB-231-bearing mice after anti-ROR1 antibodytreatment (FIG. 9K). The error bars indicate SEM; *p<0.05, **p<0.01;based on a unpaired two-sided student's t-test.

Constructs depicted in FIG. 6 were used to select high affinityrecombinant antibodies. Native western also indicated all threehumanized D10-like mAbs target the same epitope as D10 and require aminoacid 138 for binding to human ROR1 (FIG. 10). Human and chimeric ROR1-exconstructs were transfected into 293 cells. This allowed for productionof recombinant human-mouse chimeric ROR1 proteins that could be sizeseparated in a non-denaturing PAGE gel or SDS PAGE gel for immunoblotanalysis with different anti-ROR1 mAb. The results indicate that bothD10 and 99961 antibodies bind to the same region, on the C-terminus ofIg-like domain, and that D10 and 99961 can bind to ROR1 under bothdenatured and native conditions. The full human extracellular domain isprovided on the far left lane of either gel (FIG. 11). Antibody 99961has a 50× higher binding affinity for ROR1 than D10 and reduced leukemicburden more than D10 (FIG. 12). The 99961 antibody was humanized toproduce four antibodies designated 99961.1, 99961.2, 99961.3 and 99961.4

Characterization of ROR1 Antibody 99961

Assays were performed to demonstrate specific activity of 99961 againstCLL cells in human cord blood reconstituted immune deficient mice.Rag-/-γ-/- mice reconstituted with human cord blood (CB) cells so as todevelop a human immune system were injected i.p. with fresh or frozenCLL PBMC. The next day the mice were given lmg/kg 99961 or D10 orcontrol mIgG i.v. Seven days later, the CLL PBMC cells from peritonealcavity were harvested and analyzed by flow cytometry (FIG. 13A). Thedata indicate that 99961 eliminates >90% of the CLL cells and has noeffect on normal human B or T cell development (FIG. 13B, C).

Studies were also performed to demonstrate the specific activity of99961 in ROR+ primary AML. The results indicate that 99961 decreases thesurvival of primary colonies and the self renewal capabilities ofsecondary colonies (FIG. 14).

Epitope mapping of the 99961 mAb demonstrated that this epitope is onlyexpressed on various cancers and not on cord blood cell or adult humanand progenitor cells or stem cells derived from fetal liver (FIG. 15).It has also been shown that 99961 binds to leukemic cells but does notcross react with normal adult tissues (FIG. 16). The Lymphomamulti-tissue array was from Lifescan Biosciences (LS-SLYCA5) withsections from 40 lymphomas, had 5 cases where Ab9991 bound to themalignant cells. The normal multi-tissue array from Biomax (FDA999) withsections from multiple different normal tissues, showed no specificareas of binding with 99961. The immunohistochemistry was performedusing heat induced antigen retrieval with high pH buffer from DAKO(Carpenteria, Calif.) followed by enhancement using biotinyl tyramideamplification (CSA kit from DAKO).

PK studies of 99961 were performed with 1 mg/mouse antibody injected ivto in Rag-/-γ-/- mice. Blood was drawn at different time points andlevels of 99961 mAb in plasma were measured by ELISA. The resultsindicate that the antibody half-life was 11.4 days, volume was 1.18 mL(47 mL/kg) and clearance was 0.072 mL/day (0.12 mL/hr/kg) all consistentwith other macromolecules and clinically utilized antibodies (FIG. 17).

Example 7 ROR1 Peptide Vaccine

As discussed above, it has been shown that D10 binds at the carboxyterminus of the Ig-like domain that is contiguous to the CRD domain ofROR1. Antibody 4A5 binds to a different epitope in the Ig-like domainand lacks biologic activity. The epitopes of the mAbs were confirmed bychimeric ROR1-ex and site-mutation of the different amino acids betweenhuman and mouse ROR1. Peptides corresponding to the extracellular domainof ROR1 where D10, 4A5 and other ROR1 antibodies bind were constructed,A19, R22 and K19. The A19 peptide corresponds to the epitope recognizedby the 4A5 mAb; R22 peptide corresponds to the epitope recognized by theD10 mAb, the 99961 mAb (i.e. VATNGKEVVSSTGVLFVKFGPC), and the humanized99961 mAbs; and K19 peptide corresponds to a region in the Kringledomain that is recognized by other mAb specific for ROR1 (FIG. 18). Thethree peptides were each conjugated at the C-terminus with keyholelimpet hemocyanin (KLH) for immunization in adjuvant complete Freund'sadjuvant (CFA) or incomplete Freund's adjuvant (IFA). A cysteine (C) wasadded at the C-terminus and used for conjugation to KLH with MBS (FIG.20). The conjugation reaction is depicted in FIG. 19. The conjugatedpeptides were shown to bind to D10 and 99961 (FIG. 21). C57BL/6 andtransgenic mice were immunized with the conjugated peptides. Antibodytiters were collected 4 weeks after immunization. R22-KLH vaccineinduced the highest titers of anti-ROR1 antisera in either C57BL/6 miceor ROR1-Tg mice (FIG. 28). This experiment was repeated with a 16 aminoacid peptide of the D10 epitope, R16 which also induced antibodies thatreacted with the human ROR1 protein, although titers were generallylower than those induced by R22-KLH (data not shown).

The anti-ROR1 antibodies induced by R22-KLH vaccine were shown to bindto surface ROR1 present on EW36, JeKo-1, or CLL cells (FIG. 29). Forthis study, a dilution of antisera from mice immunized with R22-KLH wereincubated with the cells for 20 minutes at 4° C. The cells then werewashed and then labeled with a goat anti-mouse Ig that was conjugatedwith a fluorochrome for detection by flow cytometry. The open histogramsare the cells stained with the goat anti-mouse Ig without firstincubating the cells with the R22-KLH antisera. The shaded histogramsare the fluorescence of cells that first were incubated with theanti-R22-KLH antisera. The increase in fluorescence of the cells is dueto the mouse anti-ROR1 antibodies bound to the surface, which then weredetected with the goat anti-mouse Ig. The pre-immunization antisera ofthese mice or the antisera of mice immunized with KLH did not bind tothese cells (FIG. 29)

The R22-KLH induced antisera was tested for complement dependentcytotoxicity. EW36, Jeko-1, CLL-1 and CLL-2 cells were washed and platedat 25 μl with 5×10⁵ cells per well in RPMI/10% FBS in round-bottom96-well plates (Corning Costar). The diluted antisera (25 μl) and 25 μlof a 1:5 dilution of baby rabbit complement were added per well. D10 mAbwas used as a positive control. All conditions were performed intriplicate. Plates were incubated for 4h at 37° C., and cells wereimmediately quantitated for viability by DiOC6/PI staining and FlowCytometric Analysis. This study indicates that either D10 or theantisera generated against the R22 peptide could directcomplement-mediated lysis of cells bearing human ROR1 (FIG. 30). Cellsthat did not bear ROR1 were not killed.

The Ig sub-classes of the antibodies induced by R22-KLH were examined.For this, we used an ELISA using plates coated with human ROR1, whichthen were incubated with diluted antisera, washed and then detectedusing enzyme-conjugated secondary antibodies specific for each of theIgG subclasses, as indicated on the x axis. The results showed thatIgG1, IgG2a, IgG2b and IgG3 were all induced in varying degrees. IgG2a,IgG2b and IgG3 are associated with Th1 profile and IgG1 is associatedwith Th2 profile. These results indicate that Th1 and Th2 CD4+ T helpercells are both activated after vaccination.

R22-KLH was used to immunize C57BL/6 mice as shown in FIG. 31. The firstinjection of KLH or R22-KLH peptide was in CFA. The second andsubsequent injections were in IFA. The animals were bled on the daysmarked with the purple arrow. Forty four days after the day of the firstinjection, the C57BL/6 mice were challenged with human-ROR1-expressingCLL cells that originated in a human ROR1-transgenic mouse. This mousewas transgenic for the T-cell-leukemia 1 (TCL1 gene). Both transgenesare under the control of a B-cell specific promoter/enhancer (E-Cμ).This leukemia resembles human CLL and expresses human surface ROR1.

The collected antisera produced a significant reduction in the leukemiacell burden in mice immunized with R22-KLH, but not in mice immunizedwith KLH. (FIG. 32)

C57BL/6 Mice

R22-KLH was used to immunize C57BL/6 mice according to the schema asshown in FIG. 33. The first injection of KLH or R22-KLH peptide was inCFA. The second and subsequent injections were in IFA. The animals werebled on the days marked with the purple arrow. Forty four days after theday of the first injection, the C57BL/6 mice were challenged withhuman-ROR1-expressing CLL cells that originated in a humanROR1-transgenic mouse that also was transgenic for the T-cell-leukemia 1(TCL1 gene). Both transgenes are under the control of a B-cell specificpromoter/enhancer (E-Cμ). This leukemia resembles human CLL andexpresses human surface ROR1.

Antibody response to human ROR1 observed in mice immunized with R22-KLHat day 42, but not in mice immunized with KLH. All 4 mice immunized withR22-KLH generated high-titer antibodies against human ROR1 as detectedvia ELISA using plates coated with the extra-cellular domain ofrecombinant human ROR1 protein. These data indicate that immunizationwith the R22-KLH peptide can break self-tolerance to ROR1, which isexpressed on all B cells of these ROR1-Tg mice. The spleens from themice given the R22-KLH peptide remained similar to control animals, butthe KLH mice had significantly larger spleens (FIG. 34).

Flow cytometry of splenocytes from C57BL/6 mice immunized with eitherKLH or R22-KLH, using flurochrome-conjugated mAb specific for CD5 orROR1. The mAb used to stain the cells binds to a non-crossblockingepitope of ROR1 than the antibodies induced by R22-KLH. Note that thereare much fewer, if any, leukemia cells in the spleens of mice immunizedwith the R22-KLH vaccine (FIG. 39).

The total number of leukemia cells found in the spleens of C57BL/6 miceinjected with R22-KLH peptide 30 days earlier with 1×10⁵ human-ROR1+ CLLcells was significantly lower than the spleens of mice injected withKLH. The number of leukemia cells per spleen was derived by multiplyingthe percent of leukemia cells in the splenocyte populations (as assessedvia flow cytometry) by the number of splenocytes harvested from thespleen (FIG. 34).

The number of CD8+ cells in the spleens of mice immunized with KLH orR22-KLH was determined by flow cytometry. Following immunization withR22-KLH there were dramatic increases in CD8 T cells, which were notincreased in mice immunized with KLH. The bottom row indicate theabsolute number of CD8 T cells harvested from the spleens of mice on day75 (FIG. 37)

C57BL/6 ROR1 Transgenic Mice

Transgenic mice were injected with either R22-KLH or KLH as shown inFIG. 38. The mice are transgenic for human ROR1 under a B-cell specificpromoter/enhancer (E-Cμ). The first injection of KLH or R22-KLH peptidewas in CFA. The second and subsequent injections were in IFA. Theanimals were bled on the days marked with the purple arrow. Forty fourdays after the day of the first injection, the C57BL/6 mice werechallenged with human-ROR1-expressing CLL cells that originated in aROR1-Tg mouse that also was transgenic for the T-cell-leukemia 1 (TCL1gene). Both transgenes are under the control of a B-cell specificpromoter/enhancer (E-Cμ). Hence these ROR1-Tg mice have B cells thatexpress human ROR1. The results demonstrate that the R22-KLH peptide caninduce anti-ROR1protective immunity in mice that express ROR1 and hencebreak self-tolerance.

Antibody response to human ROR1 was observed in ROR1-Tg mice immunizedwith R22-KLH at day 42, but not in mice immunized with KLH. All 4 miceimmunized with R22-KLH generated high-titer antibodies against humanROR1 as detected via ELISA using plates coated with the extra-cellulardomain of recombinant human ROR1 protein. Further analysis by flowcytometry demonstrated that there are fewer, if any, leukemia cells inthe spleens of mice immunized with the R22-KLH vaccine than miceimmunized with KLH (FIG. 40). FACs analysis also showed that ROR1 wasdown modulated in the mice immunized with R22-KLH but not the miceimmunized with KLH. Spleens from mice immunized with R22-KLH hadsignificantly fewer leukemia cells compared to mice immunized with KLH.As with the C57BL/6 mice, immunization with R22-peptide-KLH led todramatic increases in CD8 T cells, which were not increased in miceimmunized with KLH (FIG. 39). Similar results were seen with CD4+ Tcells (FIG. 43) and CD3+ T cells (FIG. 42).

BALB/c Mice

BALB/c mice were immunized with KLH or R22-KLH as shown in FIG. 22.

For this, KLH or KLH-conjugated peptide each was formed into an emulsionwith adjuvant (CFA or IFA). CFA was used for the first immunization andIFA was used for the subsequent boost. The bleeding and peptideinjection days are indicated.

R22-KLH induced anti-ROR1 antibody levels were determined by ELISA.Purified ROR1-extacellular domain was coated to 96-well plate andincubated anti-sera with indicated dilution times from individualbleeding days. ELISA results indicated that the concentrations ofanti-ROR1 antibodies were induced in immunized BALB/c mice over time.The sera from these animals collected prior to immunization did notreact with the ROR1 protein, even at low serum dilution.

Immunoblot analysis also indicated that anti-ROR1 antibodies generatedby R22-KLH immunization of BALB/c mice produced anti-ROR1 antibodiesthat had the same epitope specificity as D10 (FIG. 23). In addition, itappears that the antisera also react with the mouse protein.

FACS analysis was confirmed the binding of anti-sera from R22-KLHimmunized BALB/c mice to ROR1 on the surface of cells.

Transgenic Mice II

Transgenic mice were immunized with either KLH or R22-KLH as shown inFIG. 24. The KLH conjugated peptide was mixed with adjuvant (CFA orIFA). CFA was used for the first immunization and IFA was used for thefollowing boost. ELISA results indicated that the concentrations ofanti-ROR1 antibodies were induced in R22-KLH immunized ROR1 transgenicmice over time. FACS analysis confirmed the binding of anti-sera fromR22-KLH immunized ROR1 transgenic mice to ROR1 on the surface of cells.

Antisera from R22-KLH immunized mice were examined for ROR1 receptorinternalization ability. MDA-MB-231 cells were incubated with anti-serafrom transgenic mice at 4° C. or 37° C. for 1 h and then stained withisotype-Alexa647, or 4A5-Alexa647 for 30 min on ice prior to FACSanalysis of ROR1 expression. The results showed that Anti-ROR1 sera fromtransgenic mice immunized with R22-KLH induced ROR1 receptorinternalization (FIG. 25)

Antisera from R22-KLH immunized mice were examined to determine theiraffect in breast cancer migration. Migrated cells were observed under10× magnification after 1 h of anti-sera treatment and then 16 h ofincubation at 37° C. Results are means±s.e.m. n=3. **p<0.01. The resultsindicated that Anti-ROR1 sera from transgenic mice could decrease breastcancer migration in vitro (FIG. 26).

1. An isolated anti-ROR1 antibody having the same binding specificity asantibody
 99961. 2. The antibody according to claim 1, wherein theantibody binds to amino acids 130-160 of hROR-1.
 3. The antibodyaccording to claim 2, wherein the antibody requires that ROR1 amino acid138 is glutamic acid for binding to hROR-1.
 4. The antibody according toclaim 1, wherein the antibody comprises a heavy chain variable region isselected from the group consisting of SEQ ID. NO:1, SEQ ID. NO:5, SEQID. NO:9, SEQ ID. NO:13 and SEQ ID. NO:17, and the light chain variableregion is selected from the group consisting of SEQ ID. NO:3, SEQ ID.NO:7, SEQ ID. NO:11, SEQ ID. NO:15 and SEQ ID. NO:19.
 5. The antibodyaccording to claim 1, wherein the antibody with a binding affinity isbetween about 500 pM and about 6 nM.
 6. The antibody according to claim5, wherein the binding affinity is about 800 pM.
 7. The antibodyaccording to claim 1, wherein the antibody is selected from the groupconsisting of 99961, 99961.1, 99961.2, 99961.3 and 99961.4.
 8. Theantibody according to claim 1, wherein the antibody inhibits metastasis.9. The antibody according to claim 1, wherein the antibody is human,humanized or chimeric.
 10. An isolated nucleic acid encoding theantibody according to claim
 1. 11. A vaccine against ROR-1 expressingcells, the vaccine comprising a pharmaceutically acceptable compositionof an isolated or synthetically produced peptide having an amino acidsequence with at least 95% sequence identity to the ROR-1 binding regionof antibody D10.
 12. The vaccine according to claim 11, wherein theamino acid sequence of the ROR-1 binding region of antibody D10 isVATNGKEVVSSTGVLFVKFGPC.
 13. The vaccine according to claim 11, whereinthe amino acid sequence of the ROR-1 binding region of antibody D10 isEVVSSTGVLFVKFGPC.
 14. The vaccine according to claim 12, wherein theROR-1 expressing cell is a cancer cell.
 15. The vaccine according toclaim 11, wherein the cancer cell is selected from the group consistingof: B cell leukemia, lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon,lung, skin, pancreatic, testicular, bladder, uterine, prostate, andadrenal cancer.
 16. The vaccine according to claim 12, furthercomprising an immunogenic adjuvant.
 17. The vaccine according to claim16, wherein the adjuvant is an immunogenic carrier peptide conjugated tothe binding peptide.
 18. The vaccine according to claim 17, wherein theamino acid sequence of the binding peptide is VATNGKEVVSSTGVLFVKFGPC andthe immunogenic carrier peptide is keyhole limpet hemocyanin (KLH),bovine serum albumin or ovalbumin.
 19. The vaccine according to claim17, wherein the amino acid sequence of the binding peptide isEVVSSTGVLFVKFGPC and the immunogenic carrier peptide is keyhole limpethemocyanin (KLH).
 20. A ROR1 binding peptide having an amino acidsequence with at least 95% sequence identity to VATNGKEVVSSTGVLFVKFGPC.21. The binding peptide according to claim 20, wherein the peptide is ofmammalian origin.
 22. An isolated nucleic acid encoding the bindingpeptide according to claim
 20. 23. A ROR1 binding peptide having anamino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC.
 24. The binding peptide according to claim 23, whereinthe peptide is mammalian.
 25. An isolated nucleic acid encoding thebinding peptide according to claim
 23. 26. A method of suppressingmetastasis of ROR-1 expressing cancer, the method comprising disruptingepithelial-mesenchymal transition of tumor cells by administering anantibody having the binding specificity of monoclonal antibody 99961, avaccine comprised of a peptide having an amino acid sequence with atleast 95% sequence identity to the ROR-1 binding region of antibody D10,a ROR-1 binding peptide having an amino acid sequence with at least 95%sequence identity to VATNGKEVVSSTGVLFVKFGPC or a ROR-1 binding peptidehaving an amino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC.
 27. The method according to claim 26, wherein theROR-1 expressing cancer is selected from the group consisting of: B cellleukemia, lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, and adrenal cancer.28. A method for treating or preventing a cancer in a subject, themethod comprising administering to the subject an antibody having thebinding specificity of monoclonal antibody 99961, a vaccine comprised ofa peptide having an amino acid sequence with at least 95% sequenceidentity to the ROR-1 binding region of antibody D10, a ROR-1 bindingpeptide having an amino acid sequence with at least 95% sequenceidentity to VATNGKEVVSSTGVLFVKFGPC or a ROR-1 binding peptide having anamino acid sequence with at least 95% sequence identity toEVVSSTGVLFVKFGPC.
 29. The method according to claim 27, wherein thecancer is selected from the group consisting of: B cell leukemia,lymphoma, CLL, AML, B-ALL, T-ALL, ovarian, colon, lung, skin,pancreatic, testicular, bladder, uterine, prostate, and adrenal cancer.